Production of beta-phellandrene using genetically engineered photosynthetic microorganisms

ABSTRACT

The present invention provides methods and compositions for producing β-phellandrene hydrocarbons from a photosynthetic microorganism such as cyanobacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/888,939, filed Feb. 5, 2018, which is a divisional of U.S.application Ser. No. 14/376,392, filed Aug. 1, 2014, which is a NationalStage of International Application No. PCT/US2013/024908, filed Feb. 6,2013, and which claims the benefit to U.S. Provisional Application No.61/595,610, filed Feb. 6, 2012, each of which is herein incorporated byreference for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS AN ASCII TEXT FILE

This application includes a Sequence Listing submitted as a text filenamed “086540-1178882-SEQ.txt” created Feb. 18, 2020, and containing50,607 bytes. The material contained in this text file is incorporatedby reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

There is a need to develop renewable biofuels and chemicals that willhelp meet global demands for energy and synthetic chemistry feedstock,but without contributing to climate change or other environmentaldegradation.

Terpenoids represent the largest and most diverse group of naturallyoccurring organic compounds, and are all derived from the monomericisoprene five-carbon building block. More than 25,000 differentnaturally occurring terpenoids have been identified, and many have plantorigin. Terpenoids are classified into groups based on the number offive-carbon isoprene units they comprise; monoterpenes (C10),sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes(C40) and polyterpenes (greater than C40). β-Phellandrene (C₁₀H₁₆)offers example of such monoterpenes as a constituent of the essentialoils synthesized by many plant species. It has significant commercialpotential for use in the cosmetics and personal care industries, incleaning products for household and industrial use, and medicinal use.There is also potential for β-phellandrene and other monoterpenes to bedeveloped as feedstock in the synthetic chemistry and pharmaceuticalindustries, and as a renewable biofuel, where β-phellandrene itself mayserve as supplement to gasoline or oligomerization of such monoterpeneunits may generate second order fuel molecules, suitable for use assupplements to jet fuel and diesel.

A number of plant species naturally produce β-phellandrene as aconstituent of their essential oils, including lavender and grand fir.Essential oils are produced and stored in specialized organs calledglandular trichomes, which form on the surface of leaves and flowers.Essential oils are mainly composed of monoterpenes and function inchemical defence against potential herbivores. The harvesting ofessential oils from glandular trichomes, and subsequent purification ofindividual monoterpenes, such as β-phellandrene, is labour intensive andcostly with relatively limited yields. The use of microorganisms, bothphotosynthetic and non-photosynthetic, for the production of suchcommercially useful and valuable chemicals is an attractive alternativeto harvesting the product from plants.

All terpenoids are produced by two biosynthetic pathways: 1) themevalonic acid (MVA) pathway, which operates in the cytosol ofeukaryotes and archaea; and 2) the methyl-erythritol-4-phosphate (MEP)pathway, which is of prokaryotic bacterial origin and present incyanobacteria, as well as in plant and algal plastids (see, FIG. 1).Synthesis of β-phellandrene in plants is due to the presence of aβ-phellandrene synthase (β-PHLS) gene. This is a nuclear gene encoding achloroplast-localized protein that catalyzes the conversion of geranyldiphosphate (GPP) to β-phellandrene. Plant β-phellandrene synthases,encoded by the gene β-PHLS, have been cloned and characterized fromlavender, grand fir, tomato, and spruce (see, e.g., Demissie et al.,Planta, 233:685-696 (2011); Bohlmann et al., Arch. Biochem. Biophys.,368:232-243 (1994); Schilmiller et al., Proc. Nat. Acad. Sci. U.S.A.,106:10865-10870 (2009); and Keeling et al., BMC Plant Biol. 11:43-57(2011)).

Although photosynthetic microorganisms, such as microalgae andcyanobacteria utilize the MEP pathway, which generates GPP precursors,these microorganisms do not natively possess a β-phellandrene synthasegene or enzyme and thus, do not natively catalyze the conversion of GPPto (3-phellandrene. However, they do express the MEP pathway and utilizethe corresponding isoprenoid pathway enzymes for the biosynthesis of agreat variety of needed terpenoid-type molecules like carotenoids,tocopherols, phytol, sterols, hormones, among many others) (see, FIG.1). The MEP isoprenoid biosynthetic pathway (Lindberg et al., MetabEng., 12:70-79 (2010)) consumes pyruvate and glyceraldehyde-3-phosphate(G3P) as substrates, which are combined to formdeoxyxylulose-5-phosphate (DXP), as first described for Escherichia coli(Rohmer et al., Biochem. J., 295:517-524 (1993)). DXP is then convertedinto methyl-erythitol phosphate (MEP), which is subsequently modified toform hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP). HMBPP is thesubstrate required for the formation of isopentenyl pyrophosphate (IPP)and dimethylallyl pyrophosphate (DMAPP), which are terpenoid precursors.Cyanobacteria also contain an IPP isomerase that catalyzes theinter-conversion of IPP and DMAPP. In addition to reactants G3P andpyruvate, the MEP pathway consumes reducing equivalents and cellularenergy in the form of NADPH, reduced ferredoxin, CTP and ATP, ultimatelyderived from photosynthesis. For reviews, see also (Ershov et al., J.Bacteriol. 184(18):5045-51; Sharkey et al., Ann. Bot. 101(1):5-18(2002)).

Evidence in the literature shows that 15-carbon hydrophobic terpenoidhydrocarbons can be transgenically expressed in photosynthetic andfermentative microorganisms, but are trapped within the cell, where theyare synthesized, requiring dewatering of the culture, drying of thebiomass, followed by product extraction from within the cells. Forexample, the sesquiterpene β-caryophyllene was produced in a transgenicstrain of the cyanobacterium Synechocystis. However, isolation of theproduct required an extensive protocol that included treating theisolated cellular biomass with an application of achloroform:methanol:water solvent mixture to solubilize lipid bilayers,releasing all intracellular compounds, and extracting the lipophiliccomponents (Reinsvold et al., Plant 168: 848-852 (2011)).

Ten-carbon monoterpene hydrocarbon products occur in different distinctconfigurations, such as acyclic (e.g., myrcene), monocyclic (e.g.,limonene and β-phellandrene), and bicyclic molecules (e.g., pinene).Spontaneous emission of monoterpene hydrocarbons from single-celledmicroorganisms to the extracellular space depends on the chemical natureof the monoterpene, and also depends on the lipid bilayer configurationand cell wall hydrophobic barriers imposed by the microorganism. Forexample, yield of limonene production increased substantially intransgenic E. coli upon the additional heterologous expression of anefflux pump from Alcanivorax borkumensis (AcrB/AcrD/AcrFa gene product;GenBank Accession No. YP692684) in the cell, suggesting limonene productfeedback inhibition and/or toxicity to the cell.

The Lavandula angustifolia β-phellandrene synthase protein has beenover-expressed in E. coli upon transformant cell induction withisopropyl β-D-1-thiogalactopyranoside, IPTG (Demissie et al., Planta,233:685-696, 2011). However, IPTG induction in E. coli can be toxic tothe cell, causing loss of cell fitness, thereby hindering a continuousand large scale production of β-phellandrene synthase by this method.Host cell toxicity could be due to accumulation of the recombinantprotein itself and/or due to synthesis and intracellular accumulation ofthe transgenic product. The latter is one of the most common barriers inthe commercial application of synthetic biology approaches for productgeneration.

This invention in based, in part, on the discovery of nucleic acids andexpression systems that can be introduced and expressed in cyanobacteriaand enable these microorganisms to produce β-phellandrene. Suchgenetically modified cyanobacteria can be used commercially in anenclosed mass culture system to provide a source of β-phellandrene whichcan be potentially developed as feedstock in the synthetic chemistry andpharmaceutical industries. For instance, β-phellandrene may serve assupplement to gasoline or oligomerization of such monoterpene units maygenerate second order fuel molecules, suitable for use as supplements tojet fuel and diesel.

BRIEF SUMMARY OF THE INVENTION

The current invention addresses the need of generating monoterpenehydrocarbons by providing methods and composition for the generation ofβ-phellandrene hydrocarbons in photosynthetic microorganisms, e.g.cyanobacteria and microalgae. β-Phellandrene, derived entirely viaphotosynthesis, i.e., from sunlight, carbon dioxide (CO₂) and water(H₂O), could serve as renewable biofuels or feedstock in the syntheticchemistry and pharmaceutical industries.

The invention is based, in part, on the discovery of improvements to theengineering of cyanobacteria which, upon suitable modification, produce10-carbon monoterpenes, such as β-phellandrene. In one aspect, theinvention therefore provides methods and compositions for producing andharvesting β-phellandrene from cyanobacteria. Such genetically modifiedorganisms can be used commercially in an enclosed mass culture system,e.g., a photobioreactor, to provide a source of renewable fuel forinternal combustion engines or, upon on-board reformation, in fuel-celloperated engines; or to provide a source of β-phellandrene for use inchemical processes such as chemical synthesis, pharmaceuticals andperfume cosmetics.

Photosynthetic microorganisms, such as microalgae and cyanobacteria donot possess a β-phellandrene synthase gene or enzyme by which tocatalyze the formation of β-phellandrene from GPP. However, they doexpress the methyl-erythritol-4-phosphate (MEP) pathway and utilize thecorresponding isoprenoid pathway enzymes for the biosynthesis of avariety of terpenoid-type molecules. This invention provides methods andcompositions to genetically modify microorganisms to express aP-phellandrene synthase gene, e.g., a codon-optimized Lavandularangustifolia β-phellandrene synthase gene, in order to produceβ-phellandrene in cyanobacteria.

In one aspect, the invention provides a method of producingβ-phellandrene hydrocarbons in cyanobacteria, the method comprising:introducing an expression cassette that comprises a nucleic acidencoding β-phellandrene synthase into the cyanobacteria, wherein thenucleic acid encoding β-phellandrene synthase is operatively linked to aPsbA2 promoter, or other suitable promoter; and culturing thecyanobacteria under conditions in which the nucleic acid encodingβ-phellandrene synthase is expressed. In some embodiments, theexpression cassette is introduced into the PsbA2 gene locus and thePsbA2 promoter is the native cyanobacteria promoter. In someembodiments, the cyanobacteria are unicellular cyanobacteria, e.g., aSynechocystis sp or a Synechococcus sp. In alternative embodiments, thecyanobacteria are multicellular, e.g., a Gloeocapsa sp. Themulticellular cyanobacteria may be a filamentous cyanobacteria sp. suchas a Nostoc sp, an Anabaena sp, or an Arthrospira sp. In someembodiments, the nucleic acid encodes a β-phellandrene synthase that hasat least 55%, 60%, 70%, 75%, or 80% sequence identity, often at least85%, 90%, 95%, or 100% sequence identity, to SEQ ID NO:3. In someembodiments, the nucleic acid encodes a β-phellandrene synthase thatcomprises amino acid SEQ ID NO:1. In typical embodiments, the nucleicacid that encodes the β-phellandrene synthase is codon-adjusted forexpression in cyanobacteria, e.g., in some embodiments, the nucleic acidis a codon-modified variant of SEQ ID NO:2. In some embodiments, theβ-phellandrene synthase nucleic acid comprises SEQ ID NO:3, or asequence having at least 80% identity, typically at least 85% identityor 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acidsequence of SEQ ID NO:3.

In other aspects, the invention provides a cyanobacteria cell, whereinthe cyanobacteria cell comprises a heterologous nucleic acid thatencodes β-phellandrene synthase and is operably linked to a promotersuch as a PsbA2 promoter. In some embodiments, the PsbA2 promoter is anendogenous promoter. In some embodiments, the cyanobacteria areunicellular cyanobacteria, e.g., a Synechocystis sp or a Synechococcussp. In alternative embodiments, the cyanobacteria are multicellularcyanobacteria, e.g., a Gloeocapsa sp. In some embodiments, themulticellular cyanobacteria sp is a filamentous cyanobacteria sp. suchas a Nostoc sp, an Anabaena sp, or an Arthrospira sp. In someembodiments, the heterologous nucleic acid encodes a P-phellandrenesynthase and has at least 55%, 60%, 70%, 75%, or 80% sequence identity,often at least 85%, 90%, 95%, or 100% sequence identity, to the nucleicacid sequence of SEQ ID NO:3. In some embodiments, the cyanobacteriacell comprises a heterologous nucleic acid that comprises the nucleicacid sequence of SEQ ID NO:3. Preferably, the heterologous nucleic acidpresent in the cyanobacterial cell that encodes the β-phellandrenesynthase is codon-optimized for expression in cyanobacteria, e.g., insome embodiments, the nucleic acid is a codon-optimized variant of SEQID NO:2. In some embodiments, the β-phellandrene synthase nucleic acidcomprises SEQ ID NO:3, or a sequence having at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3. Theinvention additionally provides vectors comprising the nucleic acid andcyanobacterial host cells into which the nucleic acid has beenintroduced.

In a further aspect, the invention provides a nucleic acid encoding aβ-phellandrene synthase that comprises amino acid SEQ ID NO:1, where thenucleic acid is a codon-optimized variant of SEQ ID NO:2 where codonsused with an average frequency of less than 12% by Synechocystis arereplaced by more frequently used codons. In some embodiments, thenucleic acid comprises the sequence set forth in SEQ ID NO:3, or asequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO:3. The invention additionally provides vectorscomprising the nucleic acid and cyanobacterial host cells into which thenucleic acid has been introduced.

In another aspect, the invention provides a method of obtainingβ-phellandrene hydrocarbons in cyanobacteria as described herein thatexpress a heterologous β-phellandrene synthase gene, where the methodcomprises mass-culturing cyanobacteria as described herein underconditions in which the β-phellandrene synthase gene is expressed.

In another aspect, the invention provides a method of obtainingβ-phellandrene in cell culture comprising genetically modifiedcyanobacteria, wherein the photosynthetically generated β-phellandreneaccumulates as a non-miscible product floating on the top of the liquidculture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Terpenoid biosynthesis via the MEP(methylerythritol-4-phosphate) pathway in photosynthetic microorganisms,e.g. Synechocystis sp. Abbreviations used: G3P=glyceraldehyde3-phosphate; DXP=deoxyxylulose 5-phosphate; HMBPP=hydroxymethylbutenyldiphosphate; IPP=isopentenyl diphosphate; DMAPP=dimethylallyldiphosphate; GPP=geranyl diphosphate; FPP=farnesyl diphosphate;GGPP=geranylgeranyl diphosphate; β-PHLS=/β-phellandrene synthase. Solidlines represent reactions catalyzed by endogenous Synechocystis enzymes,whereas the dashed line show the reaction catalyzed by theheterologously expressed S-/β-PHLS construct.

FIG. 2. Amino acid sequence alignment of β-phellandrene synthase proteinfrom lavender (Lavandula angustifolia), SEQ ID NO:4; tomato (Solanumlycopersicum), SEQ ID NO:5; grand fir (Abies grandis), SEQ ID NO:6, andspruce (Picea sitchensis 2, 3, 1, and 4), SEQ ID NOS:7, 8, 9, and 10,respectively. The Clustal 2.1 software application (University College,Dublin, Ireland) was used to perform the multiple sequence alignmentanalysis.

FIG. 3. The β-phellandrene synthase nucleotide and protein sequencesemployed in the present invention. (Part A) Amino acid sequence ofS-β-PHLS protein (SEQ ID NO:1) catalyzing the conversion of GPP toβ-PHL. (Part B) The L. angustifolia β-PHLS (La-β-PHLS) cDNA nucleotidesequence (SEQ ID NO:2; GenBank Accession No. HQ404305). The chloroplasttransit peptide is indicated in bold, and start and stop codons areunderlined. (Part C) Codon-optimized version of Lavandula angustifoliaβ-PHLS cDNA nucleotide sequence minus the chloroplast transit peptide(SEQ ID NO:3) for expression in microorganisms, e.g. Synechocystis sp.PCC 6803 and E. coli. This codon-optimized sequence was termed S-β-PHLS.Start and stop codons are indicated. Restriction sites incorporated intothe synthesized sequence for cloning purposes are underlined; PacI andNdeI sites at the start of the sequence, and BglII and NotI sites afterthe stop codon.

FIG. 4. Plasmid construct for expression of S-β-PHLS in cyanobacteria,e.g. Synechocystis. The Synechcystis codon-optimized β-phellandrenesynthase gene (S-β-PHLS) and a chloramphenicol resistance cassette(CamR), were cloned into a vector containing upstream and downstreamregions of the Synechocystis PsbA2 gene. Restriction sites used forcloning purposes are indicated. This plasmid was used for thetransformation of wild-type Synechocystis cells, and facilitated theintegration of the S-β-PHLS-CamR cassette within the Synechocystisgenome at the PsbA2 locus via double homologous recombination.

FIG. 5. Double homologous recombination and Synechocystis DNA copysegregation. Panel A shows maps of the PsbA2 gene locus in wild-typeSynechocystis and in the S-β-PHLS transformants upon integration of theS-β-PHLS-CamR gene construct into the Synechocystis genome via doublehomologous recombination upon transformation with plasmidpBA2SynβPHLSCamRA2. Genomic PCR primers (arrows) were designed toflanking regions of the upstream and downstream regions of the PsbA2gene (PsbA2 us, PsbA2 ds) that were used for homologous recombination.These amplify a 3.6 kb product in the S-β-PHLS transformant compared toa 2.3 kb product in the wild type. Panel B shows complete DNA copysegregation in 12 transformant lines following the replacement of PsbA2with the heterologous S-β-PHLS transgene construct using theabove-mentioned primers. A PCR product of ˜2.3 kb was amplified in thewild type (WT) containing the endogenous PsbA2, whereas larger productsof ˜3.6 kb were amplified in twelve different S-β-PHLS transformantlines (1-12). Absence of the 2.3 kb product from the latter indicateshomoplasmy for the introduced transgene. M, 1 kb plus marker.

FIG. 6. Western blot analysis of the S-β-PHLS protein in transformantSynechocystis cells. (A) Western blot analysis of wild type (WT) andS-β-PHLS transformant cells probed with β-PHLS specific polyclonalantibodies. Lanes were loaded with a total cell extract (TCE) sample, orthe soluble fraction of Synechocystis cells (SP) as obtained bycollection of the supernatant following cell disruption andcentrifugation to pellet insoluble material. (B) Coomassie-stainedSDS-PAGE gel corresponding to the protein profile of the Western blot inpanel A, shown as a control for protein loading.

FIG. 7. GC-MS analyses of gases from the headspace of wild type culture.Accumulated headspace gases in sealed cultures were analyzed by GC-MSfollowing 48 h of photoautotrophic growth in the presence of CO₂ ingaseous/aqueous two-phase bioreactors. GC profile of gasses fromwild-type culture.

FIG. 8. GC-MS analyses of gases from the headspace of S-β-PHLStransformant culture. Accumulated headspace gases in sealed cultureswere analyzed by GC-MS following 48 h of photoautotrophic growth in thepresence of CO₂ in gaseous/aqueous two-phase bioreactors. GC profile ofgasses from S-β-PHLS transformant culture. The β-phellandrene peak islabeled with asterisks and has a retention time of around 4.6 min.

FIG. 9. GC profile of gasses from a vaporized α-phellandrene standard(containing β-phellandrene as a contaminant). The β-phellandrene peak islabeled with asterisks and has a retention time of around 4.6 min.

FIG. 10. GC-MS analyses of gases from the headspace of an S-β-PHLSculture. Accumulated headspace gases in sealed cultures were analyzed byGC-MS following 48 h of photoautotrophic growth in the presence of CO₂in gaseous/aqueous two-phase bioreactors. MS analysis of the productseluted at 4.6 min in the S-β-PHLS transformant culture showing thesignature [77, 91, 93 and 136] MS lines of β-phellandrene.

FIG. 11. MS analysis of the products eluted at 4.6 min with acontaminating β-phellandrene peak in the standard solution.

FIG. 12. Absorbance spectra of phellandrene hydrocarbons in heptane.(Panel A) Absorbance spectra of heptane-extracted samples from thesurface of wild type (black) and S-β-PHLS transformant (S-β-PHLS) liquidcultures. The β-phellandrene absorbance peak is observed at 230 nm,exclusively in the heptane extracts from the S-β-PHLS cultures. (PanelB) Absorbance spectra of the α-phellandrene standard diluted in heptane.The α-phellandrene absorbance peak is observed at 260 nm.

FIG. 13. Comparative photoautotrophic growth measurements of wild typeand S-β-PHLS transformants in liquid culture. Photoautotrophic growthkinetics of wild type (open squares) and four different S-β-PHLStransformant lines (closed squares, circles, diamonds and triangles), asmeasured by optical density of the culture at 730 nm. Cultures weregrown under conditions of continuous aeration and illumination at 20μmol photons m⁻² s⁻¹.

FIG. 14. Quantum yields of photosynthesis as measured by oxygenevolution in wild type and S-β-PHLS transformants in liquid culture.Light saturation curves of photosynthesis for wild type and S-β-PHLStransformant cells, as measured by the oxygen-evolution activity of analiquot of the cultures incubated in the presence of 15 mM NaHCO₃, pH7.4 under a range of actinic light intensities.

FIG. 15. Absence of β-phellandrene hydrocarbons in heptane extracts fromthe surface of Escherichia coli cultures induced by isopropylβ-D-1-thiogalactopyranoside (IPTG) and over-expressing theβ-phellandrene protein. Absorbance spectra of heptane-extracted samplesfrom the surface of E. coli liquid cultures, measured in the wavelengthregion between 200 and 400 nm. Extraction time of cultures, i.e.,application of the heptane solvent on the surface of the liquid phase ofIPTG-induced cultures, was either 1 h or 48 h. No distinctiveβ-phellandrene absorbance peak could be observed at 230 nm from theseβ-PHLS cultures, as compared to that of FIG. 12.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “β-phellandrene hydrocarbon”, “β-phellandrene” or “β-PHL” in thecontext of this invention refers to a monoterpene with a chemicalformula C₁₀H₁₆. The IUPAC name is3-Methylene-6-(1-methylethyl)cyclohexene or3-methylidene-6-propan-2-ylcyclohexene. β-phellandrene is also referredto as 3-isopropyl-6-methylene-1-cyclohexene or p-mentha-1(7),2-diene.The CAS number is 555-10-2 and Pubchem CID number is 11142.β-Phellandrene is a water-insoluble cyclic monoterpene with anendocyclic and an exocyclic double bond.

A “β-PHLS gene” or “β-PHLS polynucleotide” in the context of thisinvention refers to a nucleic acid that encodes a β-PHLS protein, orfragment thereof In some embodiments, the gene is a cDNA sequence thatencodes β-PHLS. In other embodiments, a β-PHLS gene may includesequences, such as introns, that are not present in a cDNA. In someembodiments, a “β-PHLS gene” refers to a nucleic acid sequence thatencodes a β-PHLS polypeptide, e.g., a β-PHLS polypeptide shown in FIG.2, or a homolog, fragment, or variant of a β-PHLS polypeptide shown inFIG. 2. In some embodiments, a “β-PHLS gene” encodes a β-PHLSpolypeptide having a sequence set forth in SEQ ID NO:1 or encodes ahomolog, fragment, or variant of the polypeptide of SEQ ID NO:1. In someembodiments, a “β-PHLS gene” comprises the coding region of SEQ ID NO:2or SEQ ID NO:3; or comprises a nucleic acid sequence that issubstantially similar to the β-PHLS protein coding region of SEQ ID NO:2or SEQ ID NO:3. Thus, in some embodiments, a β-PHLS polynucleotide: 1)comprises a region of about 15 to about 50, 100, 150, 200, 300, 500,1,000, 1500, or 1700 or more nucleotides, sometimes from about 20, orabout 50, to about 1800 nucleotides and sometimes from about 200 toabout 600 or about 1700 nucleotides of SEQ ID NO:2 or SEQ ID NO:3; or 2)hybridizes to SEQ ID NO:2 or SEQ ID NO:3, or the complements thereof,under stringent conditions, or 3) encodes a β-PHLS polypeptide orfragment of at least 50 contiguous amino acids, typically of at least100, 150, 200, 250, 300, 350, 400, 450, 500, or 550, or more contiguousresidues of a β-PHLS polypeptide shown in FIG. 2, such as the lavenderβ-PHLS sequence SEQ ID NO:1; or 4) encodes a β-PHLS polypeptide orfragment that has at least 25%, 30%, 35%, 40%, 45%, 45%, 50%, or 55%,and often at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater,identity to SEQ ID NO:1, or over a comparison window of at least 100,200, 300, 400, 500, or 550 amino acid residues of SEQ ID NO:1; or 5) hasa nucleic acid sequence that has greater than about 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide sequenceidentity to SEQ ID NO:2 or SEQ ID NO:3, at least 80%, 85%, 90%, or atleast 95%, 96%, 97%, 98%, 99% or greater identity over a comparisonwindow of at least about 50, 100, 200, 500, 1000, 1500, 2000, or morenucleotides of SEQ ID NO:2 or SEQ ID NO:3; or 6) is amplified by primersto SEQ ID NO:2 or SEQ ID NO:3. The term “β-PHLS polynucleotide” refersto double stranded or singled stranded nucleic acids. The β-PHLS nucleicacids for use in the invention encode an active β-PHLS that catalyzesthe conversion of geranyl diphosphate (GPP) or neryl-diphosphate (NPP),which is the cis isomer of GPP, to β-phellandrene.

A “codon-optimized variant of a β-PHLS nucleic acid”, e.g., acodon-optimized variant of SEQ ID NO:2 in the context of this invention,refers to a variant that encodes the same protein, e.g., SEQ ID NO:1,but contains nucleotide substitutions based on frequency of codonoccurrence in cyanobacteria. For instance, SEQ ID NO:3 represents acodon-optimized variant of SEQ ID NO:2 for expression in theglucose-tolerant cyanobacterial strain Synechocystis sp. PCC 6803. Themethod of generating a codon-optimized variant includes modifying one ormore codons of a gene to eliminate codons that are rarely used in thehost cell, and adjusting the AT/GC ratio to that of the host cell. Rarecodons can be defined, e.g., by using a codon usage table derived fromthe sequenced genome of the host cell.

A “β-PHLS polypeptide” as herein refers to a β-PHLS polypeptide aprotein that catalyzes the conversion of geranyl diphosphate (GPP) orneryl-diphosphate (NPP) to β-phellandrene. A “β-PHLS polypeptide” thusrefers to a polypeptide having the amino acid sequence of a β-PHLS shownin FIG. 2, or a fragment or variant thereof. In some embodiments, aβ-PHLS polypeptide has the amino acid sequence of SEQ ID NO:1, or afragment or variant thereof. Thus, a β-PHLS polypeptide can: 1) have atleast 25%, 30%, 35%, 40%, 45%, 45%, 50%, or 55%, and typically at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to SEQ IDNO:1, or over a comparison window of at least 100, 200, 250, 300, 250,400, 450, 500, or 550 amino acids of SEQ ID NO:1, or has at least 70%,75%, 80%, 85%, 90%, 95% or greater identity to a β-PHLS polypeptide ofFIG. 2; or a subfragment comprising at least 100, 200, 250, 300, 250,400, 450, 500, or 550 amino acids of a β-PHLS polypeptide of FIG. 2; or2) comprise at least 100, typically at least 200, 250, 300, 350, 400,450, 500, 550, or more contiguous amino acids of a β-PHLS shown in FIG.2, or comprise at least 100, typically at least 200, 250, 300, 350, 400,450, 500, 550, or more contiguous amino acids of SEQ ID NO:1; or 3)specifically binds to antibodies raised against an immunogen comprisingan amino acid sequence of a β-PHLS of FIG. 2, e.g., SEQ ID NO:1.

As used herein, a homolog or ortholog of a particular β-PHLS gene (e.g.,SEQ ID NO:2) is a second gene in the same plant type or in a differentplant type that is substantially identical (determined as describedbelow) to a sequence in the first gene.

In the case of expression of transgenes one of skill will recognize thatthe inserted polynucleotide sequence need not be identical and may be“substantially identical” to a sequence of the gene from which it wasderived. As explained below, these variants are specifically covered bythis term.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “β-PHLS polynucleotide sequence” or“β-PHLS gene”.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Add. APL. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (CLUSTAL, GAP, BESTFIT,BLAST, FASTA, and TFASTA), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity. A“comparison window”, as used herein, includes reference to a segment ofany one of the number of contiguous positions, e.g., 20 to 600, usuallyabout 50 to about 200, more usually about 100 to about 150 in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.

The term “substantial identity” in the context of polynucleotide oramino acid sequences means that a polynucleotide or polypeptidecomprises a sequence that has at least 50% sequence identity to areference sequence. Alternatively, percent identity can be any integerfrom 50% to 100%. Exemplary embodiments include at least: at least 25%,30%, 35%, 40%, 45%, 50%55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or 100% identity compared to a reference sequence using the programsdescribed herein; preferably BLAST using standard default parameters, asdescribed below. Accordingly, β-PHLS sequences of the invention includenucleic acid sequences that have substantial identity to thecodon-optimized version of the L. angustifolia β-PHLS coding region (SEQID NO:3) or to the L. angustifolia β-PHLS coding region (SEQ ID NO:2).As noted above, β-PHLS polypeptide sequences of the invention includepolypeptide sequences having substantial identify to SEQ ID NO:1.

The terms “nucleic acid” and “polynucleotide” are used synonymously andrefer to a single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid ofthe present invention will generally contain phosphodiester bonds,although in some cases, nucleic acid analogs may be used that may havealternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphophoroamiditelinkages (see, e.g., Eckstein, F., Oligonucleotides and Analogues: APractical Approach, Oxford University Press, 1991); and peptide nucleicacid backbones and linkages. Other analog nucleic acids include thosewith positive backbones; non-ionic backbones, and non-ribose backbones.Thus, nucleic acids or polynucleotides may also include modifiednucleotides, that permit correct read through by a polymerase.“Polynucleotide sequence” or “nucleic acid sequence” includes both thesense and antisense strands of a nucleic acid as either individualsingle strands or in a duplex. As will be appreciated by those in theart, the depiction of a single strand also defines the sequence of thecomplementary strand; thus the sequences described herein also providethe complement of the sequence. Unless otherwise indicated, a particularnucleic acid sequence also implicitly encompasses variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The nucleic acid may be DNA,both genomic and cDNA, RNA or a hybrid, where the nucleic acid maycontain combinations of deoxyribo- and ribo-nucleotides, andcombinations of bases, including uracil, adenine, thymine, cytosine,guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA, or the primaryamino acid sequence of a specific protein or peptide, or a binding sitefor a trans-acting regulatory agent. This phrase specificallyencompasses degenerate codons (i.e., different codons which encode asingle amino acid) of the native sequence or sequences that may beintroduced to conform with codon preference in a specific host cell. Inthe context of this invention, the term “β-PHLS coding region” when usedwith reference to a nucleic acid reference sequence such as SEQ ID NO:2or 3 refers to the region of the nucleic acid that encodes β-PHLSprotein.

The term “promoter” or “regulatory element” refers to a region orsequence determinants located upstream or downstream from the start oftranscription that direct transcription. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal elements, whichcan be located as much as several thousand base pairs from the startsite of transcription. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter) and a second nucleic acid sequence, such as aβ-PHLS gene, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.A “cyanobacteria promoter” is a promoter capable of initiatingtranscription in cyanobacterial cells, respectively. Such a promoter istherefore active in a cyanobacteria cell, but need not originate fromthat organism. It is understood that limited modifications can be madewithout destroying the biological function of a regulatory element andthat such limited modifications can result in cyanobacteria regulatoryelements that have substantially equivalent or enhanced function ascompared to a wild type cyanobacteria regulatory element. Thesemodifications can be deliberate, as through site-directed mutagenesis,or can be accidental such as through mutation in hosts harboring thecyanobacteria regulatory element as long as the ability to conferexpression in unicellular and multicellular cyanobacteria issubstantially retained.

An “expression construct” in the context of this invention refers to anucleic acid encoding a β-PHLS protein operably linked to a promoter.The nucleic acid encoding the β-PHLS protein is considered to beheterologous to a cyanobacterial host cell, as cyanobacteria do not havea β-PHLS. An expression construct includes embodiments in which theβ-PHLS nucleic acid is linked to an endogenous promoter, e.g., theβ-PHLS nucleic acid may be integrated into cyanobacterial DNA such thatexpression is controlled by the native promoter. In further embodiments,the β-PHLS nucleic acid is operably linked to a promoter that isintroduced into the cyanbacterial host cell with the β-PHLS.

A “PsbA2 promoter” refers to a promoter region that regulates expressionof psbA2. The promoter region the psbA2 gene has been well characterized(Eriksson et al., Mol Cell Biol Res Commun 3: 292-298 (2000); Mohamed etal., Mol Gen Genet 238: 161-168 1(993); Mohamed and Jansson, Plant MolBiol 13: 693-700 (1989)). Often, the PsbA2 promoter that is operablylinked to the β-PHLS gene of this invention is the endogenouscyanobacteria promoter, but a heterologous PsbA2 promoter may also beemployed. Such promoter sequences typically include High LightRegulatory 1 (HLR1) sequences that are involved in photoregulation aswell as minimal promoter sequences (see, e.g., Eriksson et al., Mol.Cell Biol Res. Commun. 3: 292-298 (2000)).

“Expression” of a β-PHLS gene in the context of this invention typicallyrefers introducing a β-PHLS gene into cyanobacteria cells, in which itis not normally expressed. Accordingly, an “increase” in β-PHLS activityor expression is generally determined relative to wild-typecyanobacteria that have no β-PHLS activity.

A polynucleotide sequence is “heterologous to” a second polynucleotidesequence if it originates from a foreign species, or, if from the samespecies, is modified by human action from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is different from any naturally occurring allelicvariants. A “heterologous” promoter is a promoter that is not native tothe host cell or that has been modified by human action.

Polypeptides that are “substantially similar” share sequences as notedabove except that residue positions that are not identical may differ byconservative amino acid changes. Conservative amino acid substitutionsrefer to the interchangeability of residues having similar side chains.For example, a group of amino acids having aliphatic side chains isglycine, alanine, valine, leucine, and isoleucine; a group of aminoacids having aliphatic-hydroxyl side chains is serine and threonine; agroup of amino acids having amide-containing side chains is asparagineand glutamine; a group of amino acids having aromatic side chains isphenylalanine, tyrosine, and tryptophan; a group of amino acids havingbasic side chains is lysine, arginine, and histidine; and a group ofamino acids having sulfur-containing side chains is cysteine andmethionine. Exemplary conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. The phrase “stringent hybridizationconditions” refers to conditions under which a probe will hybridize toits target subsequence, typically in a complex mixture of nucleic acid,but to no other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Probes. (Tijssen, P.,ed.), Elsevier, N.Y. (1993). Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at65° C., with wash in 0.2X SSC, and 0.1% SDS at 55° C., 60° C., or 65° C.Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.For example, P-phellandrene synthase polynucleotides, can also beidentified by their ability to hybridize under stringent conditions(e.g., Tm ˜40° C.) to nucleic acid probes having the sequence of SEQ IDNO:2 or by their ability to hybridize under stringent conditions (e.g.,Tm ˜40° C.) to nucleic acid probes having the sequence of SEQ ID NO:3.Such a P-phellandrene synthase nucleic acid sequence can have, e.g.,about 25-30% base pair mismatches or less relative to the selectednucleic acid probe. SEQ ID NOS:2 and 3 are examples of nucleic acidsthat encode a L. angustifolia β-phellandrene synthase polypeptide.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state and may be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest.

As used herein, “mass-culturing” refers to growing large quantities ofcyanobacteria, that have been modified to express a β-phellandrenesynthase gene. A “large quantity” is generally in the range of about 100liters to about 1,500,000 liters, or more. In some embodiments, theorganisms are cultured in large quantities in modular bioreactors, eachhaving a capacity of about 1,000 to about 1,000,000 liters.

A “bioreactor” in the context of this invention is any enclosedlarge-capacity vessel in which cyanobacteria are grown. A“large-capacity vessel” in the context of this invention can hold about100 liters, often about 500 liters, or about 1,000 liters to about1,000,000 liters, or more.

As used herein, “harvesting” or “isolating” β-phellandrene hydrocarbonsrefers to collecting the β-phellandrene that has diffused into culturemedium from the culture medium.

Introduction

The invention employs various routine recombinant nucleic acidtechniques. Generally, the nomenclature and the laboratory procedures inrecombinant DNA technology described below are those well-known andcommonly employed in the art. Many manuals that provide direction forperforming recombinant DNA manipulations are available, e.g., MolecularCloning, A Laboratory Manual. (Sambrook, J. and Russell, D., eds.), CSHLPress, New York (3rd Ed, 2001); and Current Protocols in MolecularBiology. (Ausubel et al., eds.), New Jersey (1994-1999).

In one aspect, the invention is based, in part, on the discovery that incyanobacteria, β-phellandrene diffuses into the culture media, unlikeother long-chain hydrocarbons of the terpenoid and fatty acidbiosynthetic pathways. Accordingly, the invention provides methods andcompositions for producing β-phellandrene by expressing β-phellandrenesynthase in cyanobacteria.

β-Phellandrene Synthase Nucleic Acids

β-phellandrene synthase nucleic acid and polypeptide sequences are knownin the art. β-phellandrene synthase genes have been isolated, sequencedand characterized from lavender (Lavandular angustifolia), grand fir(Abies grandis), tomato (Solanum lycopersicum) and spruce (Picea abies,Picea sitchensis). See, e.g., Demissie et al., Planta, 233:685-696(2011); Bohlmann et al., Arch. Biochem. Biophys., 368:232-243 (1994);Schilmiller et al., Proc. Nat. Acad. Sci. U.S.A., 106:10865-10870(2009); and Keeling et al., BMC Plant Biol. 11:43-57 (2011).Illustrative accession numbers are: lavender (Lavandula angustifoliacultivar Lady), Accession: HQ404305; tomato (Solanum lycopersicum),Accession: FJ797957; grand fir (Abies grandis), Accession: AF139205;spruce (Picea sitchensis) (4 genes identified, Accession Nos: Q426162(PsTPS-Phel-1), HQ426169 (PsTPS-Phel-2), HQ426163 (PsTPS-Phel-3),HQ426159 (PsTPS-Phel-4). FIG. 2 illustrates an amino acid alignment ofβ-phellandrene synthases from lavender, grand fir, tomato and spruce.The conserved motifs are underlined.

Amino acid sequence comparison of lavender (Lavandular angustifolia)β-phellandrene synthase with those of grand fir (Abies grandis) andtomato (Solanum lycopersicum) showed 29% and 15% identity, respectively.There is a 26% amino acid sequence identity between Lavandularangustifolia (lavender) and Picea sitchensis (spruce) β-phellandrenesynthases. In terms of similarity, amino acid sequence comparison oflavender (Lavandular angustifolia) β-phellandrene synthase with those ofgrand fir (Abies grandis) and tomato (Solanum lycopersicum) showed 75%and 61% similarity, respectively. There is 73% amino acid sequencesimilarity between Lavandular angustifolia (lavender) and Piceasitchensis (spruce) β-phellandrene synthases. Although amino acidsequence comparison of all known β-phellandrene synthases, shown in FIG.2, revealed a low amino acid identity over the length of all thesequences, there are regions that are conserved.

β-Phellandrene synthases (and other monoterpene synthases such aslinalool synthase) share several conserved motifs (see, e.g., Demissieet al., Planta 233:685-696, (2011)). FIG. 2 illustrates an amino acidalignment of β-phellandrene synthase proteins from Lavandularangustifolia, Abies grandis, Solanum lycopersicum and Picea sitchensis.The monoterpene synthase signature arginine-rich N-terminal RR(x8)Wmotif (underlined in FIG. 2) is required for cyclization ofgeranyl-diphosphate (see, e.g., Williams J G K. Methods Enzymol.,167:766-778 (1988)). The arginine rich motif is located near theN-terminus of the mature protein (Demissie et al., Planta 233:685-696,(2011)). The highly conserved aspartate-rich DDxxD motif (underlined inFIG. 2) is required for substrate binding, a process usually assisted bydivalent cations, e.g. Mg²⁺ (see, e.g., Nieuwenhuizen et al., J. Exp.Bot. 60(11):3203-3219 (2009)). The partially conserved amino acidsequences, LQLYEASFLL (SEQ ID NO:11) (underlined in FIG. 2) and(N,D)D(L,I,V)x(S,T)xxxE (underlined in FIG. 2) play roles in catalysisand second metal ion binding, respectively (see, e.g., Wise et al., J.Biol. Chem., 273:14891-14899 (1998); Degenhardt et al, Phytochemistry,70 (15-16):1621-1637 (2009).; Roeder et al., Plant Mol Biol65(1):107-124 (2007)). A β-phellandrene synthase gene for use in theinvention encodes a protein retaining the motifs. Further, one of skillcan employ an alignment of the protein sequences to select residues thatmay be varied, e.g., by conservative substitution, that retain function.

Differences between monoterpene synthases have been identified inseveral plant organisms. For instance, monoterpene synthases (e.g.,β-phellandrene synthase and linalool synthase) from a given organismhave greater homology to each other, compared to monoterpene synthaseorthologs from different species. Substrates of monoterpene synthasescan also vary between plant species. For example, tomato β-phellandrenesynthase uses neryl diphosphate (NPP), which is the cis-isomer of GPP,as a substrate, rather than geranyl-diphosphate, a common substrate forother known monoterpene synthases (Schilmiller et al., Proc. Natl. Acad.Sci. USA 106:10865-10870, 2009).

The methods of the invention comprise expressing a nucleic acid sequencethat encodes a β-phellandrene synthase polypeptide, e.g., a polypeptidehaving a sequence at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, or greater, identity to a β-PHLSpolypeptide set forth in FIG. 2, e.g., a SEQ ID NO:1, in cyanobacteria.A β-PHLS polypeptide encoded by a nucleic acid employed in the methodsof the invention have the catalytic activity of converting GPP or itscis-isomer to β-phellandrene. In some embodiments, the inventionprovides a β-PHLS gene that encodes a modified version of a β-PHLSpolypeptide from a plant, such as lavender, grand fir, tomato, orspruce. A β-PHLS polypeptide variant suitable for use in the presentinvention possesses the ability to convert GPP or NPP to β-phellandrenewhen heterologously expressed in cyanobacteria. In some embodiments, theβ-PHLS polypeptide variant employs GPP. In some embodiments, a β-PHLSfor use in the invention has at least 70%, typically at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or greater,identity to a β-PHLS polypeptide from lavender, grand fir or spruce setforth in FIG. 2. Typically, the level of activity is equivalent to theactivity exhibited by a natural β-phellandrene synthase polypeptide(e.g., SEQ ID NO:1) to produce β-phellandrene. A β-phellandrene synthasepolypeptide suitable for producing β-phellandrene has at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 95%, or greater, of the activity ofan endogenous β-PHLS polypeptide from a plant, such as lavender, grandfir, tomato, and spruce, e.g., a β-PHLS having a sequence set forth inSEQ ID NO:2.

Activity of a heterologous β-phellandrene synthase of the presentinvention can be assayed by methods known to those skilled in the art.Non-limiting examples of assays that measure the function ofβ-phellandrene synthase to produce β-phellandrene from the substrate GPPor NPP include in vitro enzymatic assays using purified recombinantβ-phellandrene synthase protein, assays that determine the enzymesaturation kinetics, GC and GC-MS analysis to measure β-phellandreneproduction (detailed description in Example), spectrophotometricanalysis for β-phellandrene quantification (detailed description inExample).

β-Phellandrene Synthase Expression Constructs

β-PHLS nucleic acid sequences of the invention are expressedrecombinantly in cyanobacteria. Expression constructs can be designedtaking into account such properties as codon usage frequencies of theorganism in which the β-PHLS nucleic acid is to be expressed. Codonusage frequencies can be tabulated using known methods (see, e.g.,Nakamura et al. Nucl. Acids Res. 28:292 (2000)). Codon usage frequencytables, including those for cyanobacteria, are also available in the art(e.g., in codon usage databases of the Department of Plant GenomeResearch, Kazusa DNA Research Institute, Japan).

In certain embodiments, the invention provides a β-PHLS gene thatencodes a L. angustifolia β-PHLS protein, where the gene is acodon-optimized variant of a lavender β-PHLS gene, e.g., acodon-modified variant of SEQ ID NO:2.

Isolation or generation of β-PHLS polynucleotide sequences can beaccomplished by well-known techniques, including amplificationtechniques and/or library screening.

Appropriate primers and probes for generating a β-PHLS gene can bedesigned based on known principles using, e.g., the β-PHLS sequencesprovided herein. For a general overview of PCR see PCR Protocols: AGuide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J.and White, T., eds.), Academic Press, San Diego (1990). An illustrativePCR for amplifying a β-PHLS nucleic acid sequence is provided in theexamples.

β-PHLS nucleic acid sequences for use in the invention include genes andgene products identified and characterized by techniques such ashybridization and/or sequence analysis using an exemplary nucleic acidsequence, e.g., SEQ ID NO:3. In some embodiments, a β-PHLS nucleic acidsequence for use in the invention has at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% to SEQ ID NO:3. In someembodiments the β-PHLS nucleic acid sequence comprises SEQ ID NO:3.

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of cyanobacteria are prepared.Techniques for transformation are well known and described in thetechnical and scientific literature. For example, a DNA sequenceencoding a β-PHLS gene (described in further detail below), can becombined with transcriptional and other regulatory sequences which willdirect the transcription of the sequence from the gene in the intendedcells of the transformed cyanobacteria. In some embodiments, anexpression vector that comprises an expression cassette that comprisesthe β-PHLS gene further comprises a promoter operably linked to theβ-PHLS gene. In other embodiments, a promoter and/or other regulatoryelements that direct transcription of the β-PHLS gene are endogenous tothe cyanobacteria and the expression cassette comprising the β-PHLS geneis introduced, e.g., by homologous recombination, such that theheterologous β-PHLS gene is operably linked to an endogenous promoterand is expression driven by the endogenous promoter.

Regulatory sequences include promoters, which may be either constitutiveor inducible. In some embodiments, a promoter can be used to directexpression of β-PHLS nucleic acids under the influence of changingenvironmental conditions. Examples of environmental conditions that mayaffect transcription by inducible promoters include anaerobicconditions, elevated temperature, or the presence of light. Promotersthat are inducible upon exposure to chemicals reagents are also used toexpress β-PHLS nucleic acids. Other useful inducible regulatory elementsinclude copper-inducible regulatory elements (Mett et al., Proc. Natl.Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717(1988)); tetracycline and chlor-tetracycline-inducible regulatoryelements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al., Mol.Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995));ecdysone inducible regulatory elements (Christopherson et al., Proc.Natl. Acad. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al.,Ecotoxicol. Environ. Safety 28:14-24 (1994)); heat shock induciblepromoters, such as those of the hsp70/dnaK genes (Takahashi et al.,Plant Physiol. 99:383-390 (1992); Yabe et al., Plant Cell Physiol.35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet. 250:533-539 (1996));and lac operon elements, which are used in combination with aconstitutively expressed lac repressor to confer, for example,IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259 (1992)).An inducible regulatory element also can be, for example, anitrate-inducible promoter, e.g., derived from the spinach nitritereductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)), or alight-inducible promoter, such as that associated with the small subunitof RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol.Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)), or alight.

In some embodiments, the promoter may be from a gene associated withphotosynthesis in the species to be transformed or another species. Forexample such a promoter from one species may be used to directexpression of a protein in transformed cyanobacteria cells. Suitablepromoters may be isolated from or synthesized based on known sequencesfrom other photosynthetic organisms. Preferred promoters are those forgenes from other photosynthetic species, or other photosyntheticorganism where the promoter is active in cyanobacteria.

In some embodiments, a promoter used to drive expression of aheterologous β-PHLS gene is a constitutive promoter. Examples ofconstitutive strong promoters for use in cyanobacteria include, forexample, the psbDI gene or the basal promoter of the psbDII gene.Various other promoters that are active in cyanobacteria are also known.These include the light inducible promoters of the psbA and psbA3 genesin cyanobacteria and promoters such as those set forth in U.S. PatentApplication Publication No. 20020164706, which is incorporated byreference. Other promoters that are operative in plants, e.g., promotersderived from plant viruses, such as the CaMV35S promoters, can also beemployed in cyanobacteria. For a description of strong and regulatedpromoters, e.g., active in the cyanobacterium Anabaena sp. strain PCC7120, see e.g., Elhai, FEMS Microbiol Lett 114:179-184, (1993)). Inother embodiments, other locus in the cyanobacterial chloroplast genomecan be used to drive expression of the heterologous β-PHLS gene,provided that the locus permits relatively high expression levels of theheterologous gene. In particular embodiments,

In some embodiments, promoters are identified by analyzing the 5′sequences of a genomic clone corresponding to a β-PHLS gene. Sequencescharacteristic of promoter sequences can be used to identify thepromoter.

A promoter can be evaluated, e.g., by testing the ability of thepromoter to drive expression in cyanobacteria in which it is desirableto introduce a β-PHLS expression construct.

A vector comprising β-PHLS nucleic acid sequences will typicallycomprise a marker gene that confers a selectable phenotype oncyanobacteria transformed with the vector. Such markers are known. Forexample, the marker may encode antibiotic resistance, such as resistanceto chloramphenicol, kanamycin, G418, bleomycin, hygromycin, and thelike.

Heterologous Expression of β-phellandrene Synthase Gene in Cyanobacteria

Cell transformation methods and selectable markers for cyanobacteria arewell known in the art (Wirth, Mol. Gen. Genet., 216(1):175-7 (1989);Koksharova, Appl. Microbiol. Biotechnol., 58(2): 123-37 (2002); Thelwellet al., Proc. Natl. Acad. Sci. U.S.A., 95:10728-10733 (1998)).Transformation methods and selectable markers for are also well known(see, e.g., Sambrook et al., supra).

The codon-optimized β-phellandrene synthase gene of the presentinvention can be expressed in any number of cyanobacteria where it isdesirable to produce β-phellandrene. Suitable unicellular cyanobacteriainclude Synechocystis sp., such as strain Synechocystis PCC 6803; andSynechococcus sp., e.g., the thermophilic Synechococcus lividus; themesophilic Synechococcus elongatus or Synechococcus 6301. Multicellular,including filamentous cyanobacteria, may also be engineered to expressβ-PHLS in accordance with this invention. Multicellularr cyanobacteriathat can be used include, e.g., Gloeocapsa, as well as filamentouscyanobacteria such as Nostoc sp., e.g., Nostoc sp. PCC 7120, Nostocsphaeroides); Anabaena sp., e.g., Anabaena variabilis; and Arthrospirasp. (“Spirulina”), such as Arthrospira platensis and Arthrospira maxima.Cyanobacteria that are genetically modified in accordance with theinvention to express a β-PHLS gene may also contain other geneticmodifications, e.g., modifications to the terpenoid pathway, to enhanceproduction of β-phellandrene.

In some embodiments, an expression construct is generated to allow theheterologous expression of the β-phellandrene synthase gene inSynechocystis through the replacement of the Synechocystis PsbA2 genewith the codon-optimized β-PHLS gene via double homologousrecombination. In some embodiments, the expression construct comprises acodon-optimized β-phellandrene synthase gene operably linked to anendogenous cyanobacteria promoter. In some aspects, the promoter is thePsbA2 promoter.

In some embodiments, cyanobacteria are transformed with an expressionvector comprising a β-PHLS gene and an antibiotic resistance gene. Adetailed description is set forth in PCT Application No.PCT/US2007/71465, which is incorporated by reference. Transformants arecultured in selective media containing an antibiotic to which anuntransformed host cell is sensitive. Cyanobacteria normally have up to100 copies of identical circular DNA chromosomes in each cell.Successful transformation with an expression vector comprising a β-PHLSgene and an antibiotic resistance gene normally occurs in only one, orjust a few, of the many cyanobacterial DNA copies. Hence, presence ofthe antibiotic is necessary to encourage expression of the transgeniccopy(ies) of the DNA for β-phellandrene production. In the absence ofthe selectable marker (antibiotic), the transgenic copy(ies) of the DNAwould be lost and replaced by wild-type copies of the DNA.

In some embodiments, cyanobacterial transformants are cultured undercontinuous selective pressure conditions (presence of antibiotic overmany generations) to achieve DNA homoplasmy in the transformed hostorganism. One of skill in the art understands that the number ofgenerations and length of time of culture varies depending on theparticular culture conditions employed. Homoplasmy can be determined,e.g., by monitoring the DNA composition in the cells to determine thepresence of wild-type copies of the cyanobacterial DNA.

“Achieving homoplasmy” refers to a quantitative replacement of most,e.g., 70% or greater, or typically all, wild-type copies of thecyanobacterial DNA in the cell with the transformant DNA copy thatcarries the β-PHLS transgene. This is normally attained over time, underthe continuous selective pressure (antibiotic) conditions applied, andentails the gradual during growth replacement of the wild-type copies ofthe DNA with the transgenic copies, until no wild-type copy of thecyanobacterial DNA is left in any of the transformant cells. Achievinghomoplasmy is typically verified by quantitative amplification methodssuch as genomic-DNA PCR using primers and/or probes specific for thewild-type copy of the cyanobacterial DNA. In some embodiments, thepresence of wild-type cyanobacterial DNA can be detected by usingprimers specific for the wild-type cyanobacterial DNA and detecting thepresence of the PsbA2 gene. Transgenic DNA is typically stable underhomoplasmy conditions and present in all copies of the cyanobacterialDNA.

In some embodiments, cyanobacterial cultures can be cultured underconditions in which the light intensity is varied. Thus, for example,when a psbA2 promoter is used as a promoter to drive β-phellandrenesynthase expression, transformed cyanobacterial cultures can be grown atlow light intensity conditions (e.g., 10-50 μmol photons m⁻² s⁻¹), thenshifted to higher light intensity conditions (e.g., 500 μmol photons m⁻²s⁻¹). The psbA2 promoter responds to the shift in light intensity byup-regulating the expression of the β-PHLS gene in Synechocystis,typically at least about 10-fold. In other embodiments, cyanobacterialcultures can be exposed to increasing light intensity conditions (e.g.,from 50 μmol photons m⁻² s⁻¹ to 2,500 μmol photons m⁻² s⁻¹)corresponding to a diurnal increase in light intensity up to fullsunlight. The psbA2 promoter responds to the gradual increase in lightintensity by up-regulating the expression of the β-PHLS gene inSynechocystis in parallel with the increase in light intensity.

Production of β-phellandrene in Cyanobacteria

Transformed cyanobacteria (transformant cyanobacteria) are grown underconditions in which the heterologous β-PHLS gene is expressed. Methodsof mass culturing cyanobacteria are known to one skilled in the art. Forexample, cyanobacteria can be grown to high cell density inphotobioreactors (see, e.g., Lee et al., Biotech. Bioengineering44:1161-1167, 1994; Chaumont, J Appl. Phycology 5:593-604, 1990).Examples of photobioreactors include cylindrical or tubular bioreactors,see, e.g., U.S. Pat. Nos. 5,958,761, 6,083,740, US Patent ApplicationPublication No. 2007/0048859; WO 2007/011343, and WO2007/098150. Highdensity photobioreactors are described in, for example, Lee, et al.,Biotech. Bioengineering 44: 1 161-1 167, 1994. Other photobioreactorssuitable for use in the invention are described, e.g., in WO/2011/034567and references cited in the background section. Photobioreactorparameters that can be optimized, automated and regulated for productionof photosynthetic organisms are further described in (Puiz (2001) ApplMicrobiol Biotechnol 57:287-293). Such parameters include, but are notlimited to, materials of construction, efficient light incidence intoreactor lumen, light path, layer thickness, oxygen released, salinityand nutrients, pH, temperature, turbulence, optical density, and thelike.

Transformed cyanobacteria that express a heterologous β-PHLS gene aregrown under mass culture conditions for the production ofβ-phellandrene. In typical embodiments, the transformed organisms aregrowth in bioreactors or fermentors that provide an enclosedenvironment. For example, in some embodiments for mass culture, thecyanobacteria are grown in enclosed reactors in quantities of at leastabout 500 liters, often of at least about 1000 liters or greater, and insome embodiments in quantities of about 1,000,000 liters or more. One ofskill understands that large-scale culture of transformed cyanobacteriathat comprise a β-phellandrene synthase gene where expression is drivenby a light sensitive promoter, such as a PsbA2 promoter, is typicallycarried out in conditions where the culture is exposed to natural light.Accordingly, in such embodiments appropriate enclosed reactors are usedthat allow light to reach the cyanobacteria culture.

Growth media for culturing cyanobacteria transformants are well known inthe art. For example, cyanobacteria may be grown on solid media such asBG-11 media (see, e.g., Rippka et al., J Gen Microbiol. 111:1-61, 1979).Alternatively, they may be grown in liquid media (see, e.g., Bentley &Melis, Biotechnol. Bioeng. 109:100-109, 2012). In typical embodimentsfor production of β-phellandrene, liquid cultures are employed. Forexample, such a liquid culture may be maintained at about 25° C. under aslow stream of constant aeration and illumination, e.g., at 20 μmolphotons m⁻² s⁻¹. In certain embodiments, an antibiotic, e.g.,chloramphenical, is added to the liquid culture. For example,chloramphenicol may be used at a concentration of 15 μg/ml.

In some embodiments, cyanobacteria transformants are grownphotoautotrophically in a gaseous/aqueous two-phase photobioreactor(see, e.g., Bentley & Melis, 2012, supra, and U.S. patent applicationno. 61/477,896). In certain embodiments, the methods of the presentinvention comprise obtaining β-phellandrene using a diffusion-basedmethod for spontaneous gas exchange in a gaseous/aqueous two-phasephotobioreactor. In particular aspects of the method, carbon dioxide isused as a feedstock for the photosynthetic generation of β-phellandrenein cell culture and the headspace of the bioreactor is filled with 100%CO₂ and sealed. This allows diffusion-based CO₂ uptake and assimilationby the cells via photosynthesis, and concomitant replacement of the CO₂in the headspace with β-phellandrene vapour and O₂. Typically, thephotosynthetically generated β-phellandrene accumulates as anon-miscible product floating on the top of the liquid culture.

In particular embodiments, a gaseous/aqueous two-phase photo-bioreactoris seeded with a culture of cyanobacterial cells and grown undercontinuous illumination, e.g., at 75 μmol photons m⁻² s⁻¹, andcontinuous bubbling with air. Inorganic carbon is delivered to theculture in the form of aliquots of 100% CO₂ gas, which is slowly bubbledthrough the bottom of the liquid culture to fill the bioreactorheadspace. Once atmospheric gases is replaced with 100% CO₂, theheadspace of the reactor is sealed and the culture is incubated, e.g.,at about 25° C. to 37° C. under continuous illumination, e.g., of 150μmol photons m⁻² s⁻¹. Slow continuous mechanical mixing is also employedto keep cells in suspension and to promote balanced cell illuminationand nutrient mixing into the liquid culture in support of photosynthesisand biomass accumulation. Uptake and assimilation of headspace CO₂ bycells is concomitantly exchanged for O₂ during photoautotrophic growth.The sealed bioreactor headspace allows for the trapping, accumulationand concentration of photosynthetically produced β-phellandrene.

In some embodiments, the photoautotrophic cell growth kinetics of thecyanobacteria transformants are similar to those of wild typecyanobacteria cells. In some embodiments, the rates of oxygenconsumption during dark respiration are about the same in wild typecyanobacteria cells. In other embodiments, the rates of oxygen evolutionand the initial slopes of photosynthesis as a function of lightintensity re comparable in wild-type Synechocystis cells andSynechocystis transformants, when both are at sub-saturating lightintensities between 0 and 250 μmol photons m⁻² s³¹ ¹.

Conditions for growing β-PHLS -expressing cyanobacteria for the purposesillustrated above are known in the art (see, e.g., the illustrativereferences cited herein). β-phellandrene hydrocarbons produced by themodified cyanobacteria can be harvested using known techniques.β-phellandrene hydrocarbons are not miscible in water and they rise toand float at the surface of the microorganism growth medium. In typicalembodiments, they are siphoned off from the surface and sequestered insuitable containers. In addition, and depending on the prevailingtemperature during the mass cultivation of the cyanobacteria,β-phellandrene can exist in vapor form above the water medium in thebioreactor container (monoterpene hydrocarbons have a relatively highboiling temperature T=170-175° C.). In some embodiments, β-phellandrenevapor is piped off the bioreactor container and condensed into liquid 1form upon cooling or low-level compression.

In typical embodiments, the photosynthetically produced β-phellandreneis in liquid form and floating on the aqueous phase of the liquidculture. In some embodiments, extraction of the β-phellandrene producedin accordance with the invention is performed by skimming the floatingβ-phellandrene from the surface of the liquid phase of the culture thatis producing the β-phellandrene and isolating β-phellandrene in pureform. In certain embodiments, photosynthetically produced non-miscibleβ-phellandrene in liquid form is extracted from the liquid phase by amethod comprising overlaying a solvent such as heptane, decane, ordodecane, on top of the liquid culture in the bioreactor, incubating forat room temperature, e.g. 30 minutes or longer; and removing thesolvent, e.g., heptane, layer containing β-phellandrene. In someembodiments, photosynthetically produced β-phellandrene is a volatileproduct accumulating in the headspace of the bioreactor used forβ-phellandrene production.

EXAMPLES

The examples described herein are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of non-critical parameters that could be changed ormodified to yield essentially similar results.

Example 1. β-Phellandrene Production Using Genetically EngineeredCyanobacteria

The invention provides method and compositions for the geneticmodification of cyanobacteria to confer upon these microorganisms theability to produce β-phellandrene (C₁₀H₁₆) upon heterologous expressionof a β-phellandrene synthase gene, e.g., a β-phellnadrene syntahse genefrom lavender (Lavandular angustifolia), grand fir (Abies grandis),tomato (Solanum lycopersicum) or spruce (Picea abies, Picea sitchensis),or a variant thereof In some embodiments, the invention provides forproduction of β-phellandrene hydrocarbons in gaseous-aqueous two-phasephotobioreactors and results in the renewable generation of ahydrocarbon bio-product, which can be used, e.g., for generating fuel,chemical synthesis, or pharmaceutical and cosmetics applications. Thisexample illustrates expression of a β-phellandrene synthase gene fromlavender in cyanobacteria to produce β-phellandrene.

This example further illustrates that β-phellandrene can becontinuously-generated in cyanobacteria transformants that express aβ-phellandrene synthase gene. Further, this example demonstrates thatβ-phellandrene can spontaneously diffuse out of cyanobacteriatransformants and into the extracellular water phase, and be collectedfrom the surface of the liquid culture as a water-floating product. Thisexample also demonstrates that this strategy for production ofβ-phellandrene alleviates product feedback inhibition, product toxicityto the cell, and the need for labor-intensive extraction protocols.

In the present example, photosynthetic microorganisms, with thecyanobacterium Synechocystis sp. PCC6803 as the model organism, weregenetically engineered to express a β-phellandrene synthase gene fromlavender (Lavandular angustifolia), thereby endowing upon them with theproperty of photosynthetic β-phellandrene production (FIG. 1).Genetically modified strains were used in an enclosed mass culturesystem to provide a renewable hydrocarbon in the form of β-phellandrenethat is suitable as biofuel or feedstock in chemical synthesis.β-Phellandrene hydrocarbon products were spontaneously emitted by thecells into the extracellular space, followed by floating to the surfaceof the liquid phase, where they were easily be collected withoutimposing any disruption to the growth/productivity of the cells. Thegenetically modified cyanobacteria remained in a continuous growthphase, constitutively generating and emitting β-phellandrene. Theexample further provides an example of a codon-optimized β-phellandrenesynthase gene for improved yield of β-phellandrene in photosyntheticcyanobacteria, e.g., Synechocystis.

Materials and Methods Strains and Growth Conditions

The E. coli strain DH5α was used for routine subcloning and plasmidpropagation, and grown in LB media with appropriate antibiotics asselectable markers at 37° C., according to standard protocols. Theglucose-tolerant cyanobacterial strain Synechocystis sp. PCC 6803(Williams, J G K. Methods Enzymol., 167:766-768 (1988)) was used as therecipient strain in this study, and is referred to as the wild type.Wild type and transformant strains were maintained on solid BG-11 mediasupplemented with 10 mM TES-NaOH (pH 8.2), 0.3% sodium thiosulfate, and5 mM glucose. Where appropriate, chloramphenicol was used at aconcentration of 15 μg/mL. Liquid cultures were grown in BG-11containing 25 mM sodium phosphate buffer, pH 7.5. Liquid cultures forinoculum purposes and for photoautotrophic growth experiments andSDS-PAGE analyses were maintained at 25° C. under a slow stream ofconstant aeration and illumination at 20 μmol photons m⁻² s⁻¹. Growthconditions employed, when measuring the production of β-phellandrenefrom Synechocystis cultures, are described below in the β-phellandreneproduction assays section.

Codon-Use Optimization of the β-phellandrene Synthase Gene forExpression in Synechocystis sp. PCC 6803 and Escherichia coli

The nucleotide and translated protein sequences of the β-phellandrenesynthase gene from Lavandula angustifolia cultivar Lady (GenBankAccession Number HQ404305) were obtained from the NCBI GenBank database(National Center for Biotechnology Information; see, e.g.,http:/lwww.ncbi.nlm.nih.gov/nuccore/HQ404305). The protein sequence ofthe β-phellandrene synthase gene was firstly analyzed by TargetPsoftware (see, e.g., http://www.cbs.dtu.dk/services/TargetP/) for theprediction of the subcellular localization of the protein and foridentification of the presence and length of any targeting/transit aminoacid sequence. Based on this analysis, the β-phellandrene synthase fromLavandula angustifolia cultivar Lady was predicted to be a chloroplastlocalized protein with the first 42 amino acids of the protein servingas a chloroplast transit peptide. This analysis indicated that the first42 amino acids are not part of the mature protein that functions in thecatalysis of GPP conversion to β-phellandrene in the chloroplast. Basedon this information, we designed a protein sequence from the originalsequence of Lavandula β-phellandrene synthase (La-β-PHLS) gene byreplacing the first 42 amino acids with a methionine. The codon-use ofthe resulting cDNA was then optimized for expression in Synechocystissp. PCC 6803 and E. coli. The protein sequence of the β-phellandrenesynthase that was employed in this work is composed of 540 amino acidsof which the sequence is shown in FIG. 3A. To maximize the expression ofβ-phellandrene synthase in Synechocystis sp. PCC 6803 and E. coli, thisprotein sequence was back-translated and codon-optimized according tothe frequency of the codon usage in Synechocystis sp. PCC 6803. Thecodon-optimization process was performed based on the codon usage tableobtained from Kazusa DNA Research Institute, Japan (see, e.g.,http://www.kazusa.or.jp/codon/), and using the “Gene Designer 2.0”software from DNA 2.0 (see, e.g., https://www.dna20.com/) at a cut-offthread of 15%. The codon-optimized gene was designed with appropriaterestriction sites flanking the S-β-PHLS sequence to aid subsequentcloning steps. The nucleotide sequences of the original β-phellandrenesynthase gene from Lavandula angustifolia (La-β-PHLS) is shown in FIG.3B, while the codon-optimized sequence for expression in Synechocystissp. PCC 6803 and E. coli (S-β-PHLS) is shown in FIG. 3C.

Plasmid Construction and Generation of Synechocystis Transformants withHeterologous Expression of the S-β-PHLS Gene

A plasmid construct was generated to allow the heterologous expressionof the β-phellandrene synthase gene in Synechocystis through thereplacement of the Synechocystis PsbA2 gene with the Syn-β-PHLS gene viadouble homologous recombination. The synthesized Syn-β-PHLS was PCRamplified using the following primers: PHLS_F,5′-CCTGGGCGGTTCTGATAACG-3′ (SEQ ID NO:12), and PHLS_BamHI_R,5′-CGCGGATCCTTTTGACGGCGGCCGCAGAT-3′ (SEQ ID NO:13). A BamHI site wasincorporated into the PHLS_BamHI_R primer to allow the cloning ofS-β-PHLS PCR product into the NdeI and BamHI sites of the plasmidpBA2A2, which contains 500 bp of the upstream and downstream sequencesof the PsbA2 gene (Lindberg et al., Metab. Eng., 12:70-79 (2010)),generating plasmid pBA2SynfβPHLSA2. Finally, a chloramphenicolresistance cassette from plasmid pACYC184 was PCR amplified usingprimers with strategically incorporated restriction sites: CamR_NotI_F,5′-AAGGAAAAAAGCGGCCGCGTTGATCGGCACGTAAGAGGTTC-3′ (SEQ ID NO:14), andCamR_BamHI_R, 5′-CGCGGATCCCCAGGCGTTTAAGGGCACCAATAAC-3′ (SEQ ID NO:15),and cloned into the NotI and BamHI sites of plasmid pBA2SynfβPHLSA2, togenerate plasmid pBA2SynfβPHLSCamRA2. This plasmid was used to transformwild-type Synechocystis sp. PCC 6803 according to established procedures(Williams J G K. Methods Enzymol., 167:766-778 (1988); Eaton-Rye J J.Methods Mol. Biol., 684:295-312 (2011)). Chloramphenicol was used forselection and maintenance of transformant strains on agar plate. Theheterologous transformed Synechocystis PCC 6803 cyanobacteria arereferred to as S-β-PHLS transformants. Successful transgeneincorporation and complete DNA cyanobacterial copy segregation for theS-β-PHLS gene was verified by genomic DNA PCR, using primers designed togenomic DNA regions just outside of the upstream and downstream regionsof the PsbA2 gene that were used for homologous recombination:

(SEQ ID NO: 16) A2us_F, 5′-TATCAGAATCCTTGCCCAGATG-3′,  and(SEQ ID NO: 17) A2ds_R, 5′-GGTAGAGTTGCGAGGGCAAT-3′.

Antibody Generation and Western Blot Analysis

For expression in E. coli, the Synechocystis codon optimized β-PHLS gene(S-β-PHLS) was PCR amplified using the forward primer5′-GGAATTCCATATGTGTAGTTTGCAAGTTTCTGAT-3′(SEQ ID NO:18) and reverseprimer 5′-ACAGGATCCTCACTCATAGCGCTCAATCAGCGT-3′ (SEQ ID NO:19), andsubcloned into the pET28a(+) vector (Novagen). Expression of theS-β-PHLS construct was induced by IPTG in E. coli BL21 (DE3) cells(Novagen), and the 6xHis-tagged S-β-PHLS protein was purified undernative conditions through a nickel-nitrilotriacetic acid agarose column(NTA, Qiagen) according to the manufacturer's instructions. Specificpolyclonal antibodies were generated in rabbit against the full lengthmature β-phellandrene synthase recombinant protein as the antigen,following the instructions of ProSci Inc, USA.

Samples for SDS-PAGE analyses were prepared from Synechocystis cellsresuspended in phosphate buffer pH 7.4 at a concentration of 0.12 mg/mlchlorophyll. The suspension was supplemented with 0.05% w/v lysozyme(Thermo Scientific) and incubated with shaking at 37° for 45 min. Cellswere then pelleted at 4,000 g, washed twice with fresh phosphate bufferand disrupted with a French Pressure chamber (Aminco, USA) at 1500 psiin the presence of 1 mM PMSF. Soluble protein was separated from thetotal cell extract by centrifugation at 21,000 g and removed as thesupernatant fraction. Samples for SDS-PAGE analysis were solubilizedwith 1 volume of 2× denaturing protein solubilization buffer (0.2 MTris, pH 6.8, 4% SDS, 2 M urea, 1 mM EDTA and 20% glycerol). Inaddition, all samples in denaturing solutions were supplemented with a5% (v/v) of β-mercaptoethanol and centrifuged at 17,900 g for 5 minprior to gel loading. For Western blot analyses, Any kD™ (BIO-RAD)precast SDS-PAGE gels were utilized to resolve proteins, which were thentransferred to PVDF membrane (Immobilon-FL 0.45 μm, Millipore,USA) forimmunodetection using the rabbit immune serum containing specificpolyclonal antibodies against the S-β-PHLS protein. Cross-reactions werevisualized by Supersignal West Pico Chemiluminiscent substrate detectionsystem (Thermo Scientific, USA).

Chlorophyll Determination, Photosynthetic Productivity and BiomassQuantitation

Chlorophyll a concentrations in cultures were determinedspectrophotometrically in 90% methanol extracts of the cells accordingto Meeks and Castenholz (Arch. Mikrobiol., 78:25-41 (1971)).Photosynthetic productivity of the cultures was tested polarographicallywith a Clark-type oxygen electrode (Rank Brothers, Cambridge, England).Cells were harvested at mid-exponential growth phase, and maintained at25° C. in BG11 containing 25 mM HEPES-NaOH, pH 7.5, at a chlorophyll aconcentration of 10 μg/mL. Oxygen evolution was measured at 25° C. inthe electrode upon yellow actinic illumination, which was defined by aCS 3-69 long wavelength pass cutoff filter (Corning, Corning, N.Y.).Photosynthetic activity of a 5 mL aliquot of culture was measured atvarying actinic light intensities in the presence of 15 mM NaHCO₃ pH7.4, to generate the light saturation curve of photosynthesis. Culturebiomass accumulation was measured gravimetrically as dry cell weight,where 5 mL samples of culture were filtered through 0.22 μm Milliporefilters and the immobilized cells dried at 90° C. for 6 h prior toweighing the dry cell weight.

β-Phellandrene Production and Quantification Assays

Synechocystis cultures for 13-phellandrene production assays were grownphotoautotrophically in 1 L gaseous/aqueous two-phase photobioreactors,described in detail by Bentley and Melis (Biotechnol Bioeng.,109:100-109 (2012)). Bioreactors were seeded with a 700 ml culture ofSynechocystis cells at an OD730 nm of 0.05 in BG11 medium containing 25mM sodium phosphate buffer, pH 7.5, and grown under continuousillumination at 75 μmol photons m⁻² s⁻¹, and continuous bubbling withair, until an OD730 nm of approximately 0.5 was reached. Inorganiccarbon was delivered to the culture in the form of 500 mL aliquots of100% CO₂ gas, which was slowly bubbled though the bottom of the liquidculture to fill the bioreactor headspace. Once atmospheric gases werereplaced with 100% CO₂, the headspace of the reactor was sealed and theculture was incubated under continuous illumination of 150 μmol photonsm⁻² s⁻¹ at 35° C. Slow continuous mechanical mixing was employed to keepcells in suspension and to promote balanced cell illumination andnutrient mixing into the liquid culture in support of photosynthesis andbiomass accumulation. Uptake and assimilation of headspace CO₂ by cellswas concomitantly exchanged for O₂ during photoautotrophic growth. Thesealed bioreactor headspace allowed for the trapping, accumulation andconcentration of photosynthetically produced β-phellandrene, as either avolatile product in the headspace, or in liquid form floating on theaqueous phase.

Gas from the headspace of sealed bioreactors was sampled and analyzed bygas chromatographymass spectrometry (GC-MS) in an effort to detectvolatilized, photosynthetically produced monoterpene hydrocarbons(β-phellandrene). Comparison of retention time and mass spectrum with avaporized mixture of α-phellandrene and β-phellandrene standard (MPBiomedicals) allowed for positive identification of β-phellandrene inthe headspace. Photosynthetically produced non-miscible β-phellandrenein liquid form was extracted from the liquid phase upon overlaying 20 mLheptane on top of the liquid culture in the bioreactor, and uponincubating for 30 min, or longer, at room temperature. The heptane layerwas subsequently removed and analysed by GC-MS for the detection ofβ-phellandrene by comparison with the liquid a-phellandrene andβ-phellandrene standard also dissolved in heptane. GC-MS analyses wereperformed with an Agilent 6890GC/5973 MSD equipped with a DB-XLB column(0.25 mm i.d.×0.25 μm×30 m, J &W Scientific). Oven temperature wasinitially maintained at 40° C. for 4 min, followed by a temperatureincrease of 5° C./min to 80° C., and a carrier gas (helium) flow rate of1.2 ml per minute.

Accumulation of β-phellandrene in the liquid phase was quantifiedspectrophotometrically according to known absorbance spectra andextinction coefficients of β-phellandrene in organic solvents (Macbethet al., J Chem. Soc. 119-123 (1938); Booker et al., J. Chem. Soc.1453-1463 (1940); Gross K P, Schnepp O., J. Chem. Phys. 68:2647-2657(1978)). The majority of photosynthetically produced β-phellandreneaccumulated as a liquid floating over the aqueous phase of thebioreactor. Therefore, the non-miscible, heptane-extractedβ-phellandrene was used to generate the absorption spectra ofβ-phellandrene in heptane for quantification purposes.

Results

The native L. angustifolia cDNA sequence has a codon usage differentfrom that preferred by photosynthetic microorganisms, e.g.,cyanobacteria and microalgae. The unicellular cyanobacteriaSynechocystis sp. were used as a model organism in the development ofthe present invention. A de novo codon-optimized β-PHLS gene wasdesigned and synthesized. In the optimized version of the gene, termedS-β-PHLS, the codon usage was adapted to eliminate codons rarely used inSynechocystis, and to adjust the AT/GC ratio to that of the host. Rarecodons were defined using a codon usage table derived from the sequencedgenome of Synechocystis. The β-phellandrene synthase sequences used inthis example were: the β-PHLS protein sequence for expression inSynechocystis and E. coli (S-β-PHLS), the native L. angustifolia β-PHLScDNA sequence including the predicted chloroplast transit peptide(GenBank Accession No. HQ404305), and the L. angustifolia β-PHLS cDNAsequence minus the chloroplast transit peptide, with codon usageoptimized for Synechocystis (S-β-PHLS). In the native L. angustifoliaβ-PHLS sequence a substantial number of codons are present that are usedwith a frequency of less than 15% by Synechocystis. In thecodon-optimized gene, such low-frequency codons were not allowed.

SDS-PAGE analyses and immuno-detection of the β-phellandrene synthaseenzyme, using specific polyclonal antibodies raised against the E.coli-expressed recombinant protein, confirmed the presence of theS-β-PHLS protein in Synechocystis (FIG. 6). The S-β-PHLS protein waslocalized in the soluble fraction of Synechocystis cell extracts,consistent with the notion of a soluble protein. FIG. 6 (top panel)shows the absence of cross-reaction between the anti-S-β-PHLS polyclonalantibodies and any protein of the Synechocysis wild type (WT) in thetotal cell extract (TCE) or supernatant (SP) fractions. However, aspecific cross-reaction was observed between the anti-S-β-PHLSpolyclonal antibodies and a protein band at about 65 kD in both of thetotal cell extract (TCE) and supernatant fractions (SP) of the S-β-PHLStransformant. These results clearly show that the recombinant S-β-PHLSprotein was expressed in Synechocystis transformants, and that itaccumulated as a soluble protein in the cell.

The above results demonstrated that Synechocystis can be used forheterologous transformation using a β-PHLS gene, and that suchtransformants expressed and accumulated the S-β-PHLS protein in theircytosol. To determine whether the expressed S-β-PHLS protein ismetabolically competent, wild type and S-β-PHLS transformants werecultivated under the conditions of the gaseous/aqueous two-phasebioreactor (Bentley F K and Melis A., Biotechnol Bioeng., 109:100-109(2012)), with 100% CO₂ gas occupying the headspace prior to sealing thereactor to allow autotrophic biomass accumulation. Samples were obtainedfrom both the headspace of sealed cultures (to detect vaporizedβ-phellandrene) and from the surface of liquid cultures (to detectnon-miscible liquid β-phellandrene floating on top of the aqueous phase)and analyzed by GC-MS.

Analysis of the accumulated reactor headspace gases in the wild typeafter 48 h incubation showed no evidence of β-phellandrene hydrocarbons(FIG. 7). The headspace of the S-β-PHLS transformant, however, showedβ-phellandrene accumulation, as evidenced by the GC peak with a 4.6 minretention time (FIG. 8, asterisk), which is comparable to the retentiontime of the β-phellandrene peak observed in the phellandrene standard(FIG. 9, asterisk). A supply of commercially available pureβ-phellandrene standard was difficult to source, therefore we opted touse a commercially-available α-phellandrene standard. Upon GC-MSanalysis of the standard, a major peak was identified as α-phellandrene,however, the MS-analysis indicated presence of a number of othermonoterpene impurities, including β-myrcene, 2-carene, benzene,eucalyptol and β-phellandrene (FIG. 9). Accordingly, we employed theβ-phellandrene peak in the standard as a reference for identification ofβ-phellandrene produced by the Synechocystis transformant cultures. Theheight of the β-phellandrene peak from the gas sample of the S-β-PHLStransformant clearly showed that β-phellandrene was the major volatilehydrocarbon generated by photosynthesis in the transformant (FIG. 8).This peak was positively identified as β-phellandrene by comparison ofits mass spectrum with the β-phellandrene peak in the standard sample,showing distinct mass spectral lines [77, 91, 93 and 136] that signifyβ-phellandrene hydrocarbons (FIGS. 10 and 11). These results providedevidence that the S-β-PHLS transgene and its encoded β-phellandrenesynthase enzyme were responsible for the catalysis of β-phellandreneproduction in the transformant Synechocystis strains.

Unlike molecular hydrogen (H₂) and isoprene hydrocarbons (C₅H₈), whichare small and easily escape from the cells that produce them (Melis A.,Energy Environ. Sci., 5(2): 5531-5539; (2012)), monoterpenehydrocarbons, including β-phellandrene, are large enough to be trappedin the hydrophobic domain of the cell's lipid bilayers. Such outcomewould most likely have very adverse consequences for cell growth andfitness. Accordingly, our efforts have focused to investigate whetherthe cells can freely emit β-phellandrene, and whether cell growth andproperties of photosynthesis are adversely affected in the S-β-PHLStransformants.

Monoterpene hydrocarbons have a relatively high boiling point (170-175°C.) and are non-miscible in aqueous solution. If freely emitted by thetransformant photosynthetic microorganisms, one would expect thatmonoterpene molecules, including β-phellandrene, would float on theaqueous phase of the reactor. Surprisingly, this was indeed observed inthe case of β-phellandrene, produced by the transformed cyanobacteria. Asmall volume of heptane was layered on top of the liquid culture to trapnon-miscible liquid hydrocarbons, such as β-phellandrene, floating onthe surface of the culture, and spectrophotometic analyses wereperformed as the method for β-phellandrene quantification. FIG. 12Ashows representative absorbance spectra of heptane-extracted samplesfrom wild type (WT) and S-β-PHLS transformant cultures. The absorbancemaximum of β-phellandrene occurs at 230 nm (Macbeth et al., J. Chem.Soc., 119-123 (1938); Booker et al., J. Chem. Soc., 1453-1463 (1940);Gross K P and Schnepp O., J. Chem. Phys., 68:2647-2657 (1978)). Awell-defined band peaking at 230 nm was observed in the heptane extractsof S-β-PHLS-transformants, while absent in wild-type samples.Importantly, α-phellandrene has an absorbance maximum of 260 nm (Macbethet al., J. Chem. Soc., 119-123 (1938); Booker et al., J. Chem. Soc.,1453-1463 (1940); Gross K P and Schnepp O., J. Chem. Phys., 68:2647-2657(1978)), which allows β-phellandrene to be easily distinguished fromα-phellandrene using the spectrophotometric method. FIG. 12B shows theabsorbance spectrum of the liquid α-phellandrene standard (used for theGC-MS analyses, e.g. FIG. 9) diluted in heptane with its absorbancemaximum of 260 nm. The absence of absorbance peaks at 260 nm in theS-β-PHLS transformants (FIG. 12A) indicated that little, if any,α-phellandrene accumulated as a non-miscible product of photosynthesisin transformant lines, and that β-phellandrene exclusively accumulatedin more substantial quantities.

Quantification of β-phellandrene in the heptane-extracted samples fromS-β-PHLS transformants was determined according to the Beer-Lambert Law,using the absorbance values measured at 230 nm and the known molarextinction coefficient of β-phellandrene. During 48 h of activephotoautotrophic growth in the presence of CO₂ in a sealedgaseous/aqueous two-phase bioreactor, a 700 ml culture of S-β-PHLStransformant produced β-phellandrene in the form of a non-miscibleproduct floating on the surface of the culture.

The photoautotrophic cell growth kinetics of the S-β-PHLS transformantswere similar to those of the wild type, with a cell doubling time of 16h under a light intensity of 20 μmol photons m⁻² s⁻¹ under continuousbubbling with air (FIG. 13). The light saturation curves ofphotosynthesis of wild type and the S-β-PHLS transformants were alsosimilar to one another (FIG. 14), where oxygen evolution saturated atabout 500 μmol photons m⁻² s⁻¹, with an average P_(max) of 216 μmol O₂(mg Chl)⁻¹h⁻¹ in wild type and 263 μmol O₂ (mg Chl)⁻¹ 11⁻¹ in theS-β-PHLS transformant (FIG. 14). Similarly, rates of oxygen consumptionduring dark respiration were about the same in the wild type andS-β-PHLS transformants and equal to about −14 μmol O₂ (mg Chl)⁻¹h⁻¹.Importantly, at sub-saturating light intensities between 0 and 250 μmolphotons m⁻² s⁻¹, rates of oxygen evolution and the initial slopes ofphotosynthesis as a function of light intensity were comparable inwild-type and S-β-PHLS-transformant cells (FIG. 14), suggesting similarquantum yields of photosynthesis for the two strains (Melis A., PlantScience, 177:272-280 (2009)). These results demonstrated that deletionof the endogenous PsbA2 coding region from the Synechocystis genome,with the attendant replacement/integration and expression of theS-β-PHLS transgene in the cell, as well as the subsequent generation andaccumulation of β-phellandrene, had no adverse effects on thephotoautotrophic growth parameters of the transformants.

The β-phellandrene synthase protein has been successfully over-expressedin E. coli (Demissie et al., Planta, 233:685-696 (2011)). However, onlyin vitro enzymatic assays were performed with the β-phellandrenesynthase recombinant protein. This suggests that there was littleβ-phellandrene in E. coli and/or limited or no efflux of β-phellandrenefrom E. coli and/or adverse effects of the β-phellandrene synthaseprotein or of its product on the E. coli host cells. Absence ofβ-phellandrene hydrocarbons in heptane extracts from the surface ofIPTG-induced β-PHLS-transformed Escherichia coli cultures was alsoobserved in the illustrative experiments described herein (FIG. 15). Inthis study, transformant E. coli cells were induced by isopropylβ-D-1-thiogalactopyranoside (IPTG), resulting in the over-expression ofthe β-phellandrene protein. Absorbance spectra of heptane-extractedsamples from the surface of such E. coli liquid cultures failed to showthe presence of the β-phellandrene molecule. No distinctiveβ-phellandrene absorbance peak could be observed at 230 nm from theβ-PHLS E. coli cultures (compare with the results of FIG. 12A).

Discussion

“Photosynthetic biofuels”, as defined in the present invention, areproduced in a system where the same organism serves both asphoto-catalyst and producer of ready-made fuel or chemical. A number ofguiding principles have been applied in the endeavor of photosyntheticbiofuels, as they pertain to the selection of organisms and,independently, to the selection of potential biofuels. Criteria for theselection of organisms include, foremost, the solar-to-biofuel energyconversion efficiency, which must be as high as possible. This importantcriterion is better satisfied with photosynthetic microorganisms thanwith crop plants (Melis A., Plant Science, 177:272-280 (2009)). Criteriafor the selection of potential biofuels include (i) the relative energycontent and potential utility of the molecule. Pure hydrocarbons arepreferred over sugars or alcohols because of the greater relative energystored in hydrocarbon molecules (Schakel et al., J. Food Comp. Anal.,10:102-114 (1997); Berg J, Tymoczko J L, Stryer L. (2002) Biochemistry(5th ed.). W. H. Freeman, San Francisco, Calif. p.603.); and (ii) thequestion of product separation from the biomass, which entersprominently in the economics of the process and is a most importantaspect in commercial application. This example demonstrates thatβ-phellandrene is suitable in this respect, as it is not miscible inwater, spontaneously separating from the biomass and end-up floating onthe aqueous phase of the reactor and culture that produced them. Suchspontaneous product separation from the liquid culture alleviates therequirement of time-consuming, expensive, and technologically complexbiomass dewatering (Danquah et al., J Chem Tech. Biotech., 84:1078-1083(2009); Saveyn et al., J. Res. Sci Tech., 6:51-56 (2009)) and productexcision from the cells that otherwise would be needed for productisolation.

In the pursuit of renewable biofuels, photosynthesis, cyanobacteria ormicroalgae and β-phellandrene meet the above-enumerated criteria for“process”, “organism” and “product”, respectively. This example showsthat β-phellandrene can be heterologously produced via photosynthesis inmicroorganisms, e.g., cyanobacteria, genetically engineered to express aplant β-phellandrene synthase. In this example, the Lavandularangustifolia β-PHLS gene was employed via heterologous expression inSynechocystis. The DNA sequence of the Lavandular β-PHLS gene wasoptimized for Synechocystis codon-usage. A diffusion-based method forspontaneous gas exchange in gaseous/aqueous two-phase photobioreactorswas employed, using carbon dioxide as a feedstock for the photosyntheticgeneration of β-phellandrene. The headspace of the bioreactor was filledwith 100% CO₂ and sealed, allowing the diffusion-based CO₂ uptake andassimilation by the cells via photosynthesis, and the concomitantreplacement of the CO₂ in the headspace with β-phellandrene vapour andO₂. A considerable amount of photosynthetically generated β-phellandreneaccumulated as a non-miscible product floating on the top of the liquidculture, which is explained as β-phellandrene has a boiling point of171° C.

In the plasmid constructs employed for the expression of theβ-phellandrene synthase in Synechocystis, we used the PsbA2 gene locusfor insertion of the transgenes. Upon transformation of Synechocystiswith these constructs, the β-PHLS gene replaced the coding sequence ofthe PsbA2 gene, and the PsbA2 promoter was used to drive expression ofβ-PHLS. The PsbA2 gene is one of three homologous genes incyanobacteria, the other two being PsbA1 and PsbA3, that encode the 32kD/D1 reaction center protein of photosystem-II. The promoter region andregulation of expression of the PsbA2 gene has been characterized(Eriksson et al., Mol. Cell Biol. Res. Commun., 3:292-8 (2000);Mohamedet al., Mol Gen Genet., 238:161-8 (1993); Mohamed A, Jansson C.,Plant Mol. Biol. 13:693-700 (1989)). It has also been shown that aknock-out mutant of either PsbA2 or PsbA3 is able to growphotoautotrophically, provided that the other PsbA genes are stillactive, while PsbA1 on its own was not able to compensate for the lossof both PsbA2 and PsbA3 (Mohamed A, Jansson C., Plant Mol. Biol.13:693-700 (1989)). Inactivation of PsbA2 resulted in a strongup-regulation of PsbA3 (Mohamedet al., Mol Gen Genet., 238:161-8(1993)). This example illustrates that replacement of PsbA2 by thecodon-optimized β-PHLS gene does not significantly alter normalphotoautotrophic growth of the transformants.

The monoterpene β-phellandrene is an energy rich 10-carbon hydrocarbonmolecule, useful industrially as in cosmetics industry, cleaningproducts for household and industrial use, and medicinal use. Currently,β-phellandrene for use in commercial industry is extracted from plants,such as lavender, which contain β-phellandrene in their glandulartrichome essential oils. However, this example shows that β-phellandrenecan be produced by photosynthetic microorganisms, e.g., cyanobacteriaand microalgae, through heterologous expression of the gene encoding forthe β-phellandrene synthase (β-PHLS), in a reaction of the MEP pathway,driven by the process of cellular photosynthesis. Since the carbon atomsused to generate β-phellandrene in such a system originate from CO₂,this would make cyanobacterial and microalgal β-phellandrene productiona carbon-neutral source of synthetic chemistry and biofuel feedstock.β-Phellandrene would also be suitable as a building block for theproduction of longer chain hydrocarbons, to be used as longer chainrenewable and carbon-neutral biofuels, pharmaceuticals, and cosmetics.

All publications, accession numbers, and patent applications cited inthis specification are herein incorporated by reference as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Illustrative Sequences

Amino acid sequence of the mature Sβ-PHLS protein SEQ ID NO: 1MCSLQVSDPIPTGRRSGGYPPALWDFDTIQSLNTEYKGERHMRREEDLIGQVREMLVHEVEDPTPQLEFIDDLHKLGISCHFENEILQILKSIYLNQNYKRDLYSTSLAFRLLRQYGFILPQEVEDCFKNEEGTDFKPSFGRDIKGLLQLYEASFLSRKGEETLQLAREFATKILQKEVDEREFATKMEFPSHWTVQMPNARPFIDAYRRRPDMNPVVLELAILDTNIVQAQFQEELKETSRWWESTGIVQELPFVRDRIVEGYFWTIGVTQRREHGYERIMTAKVIALVTCLDDIYDVYGTIEELQLFTSTIQRWDLESMKQLPTYMQVSFLALHNEVTEVAYDTLKKKGYNSTPYLRKTWVDLVESYIKEATWYYNGYKPSMQEYLNNAWISVGSMAILNHLFFRFTNERMHKYRDMNRVSSNIVRLADDMGTSLAEVERGDVPKAIQCYMNETNASEEEAREYVRRVIQEEWEKLNTELMRDDDDDDDFTLSKYYCEVVANLTRMAQFIYQDGSDGFGMKDSKVNRLLKETLIERYEThe native La-β-PHLS cDNA nucleotide sequence SEQ ID NO: 2ATGTCTACCATTATTGCGATACAAGTGTTGCTTCCTATTCCAACTACTAAAACATACCCTAGTCATGACTTGGAGAAGTCCTCTTCGCGGTGTCGCTCCTCCTCCACTCCTCGCCCTAGACTGTGTTGCTCGTTGCAGGTGAGTGATCCGATCCCAACGGGCCGGCGATCCGGAGGCTACCCGCCCGCCCTATGGGATTTCGACACTATTCAATCGCTCAACACCGAGTATAAGGGAGAGAGGCACATGAGAAGGGAAGAAGACCTAATTGGGCAAGTTAGAGAGATGCTGGTGCATGAAGTAGAGGATCCCACTCCACAGCTGGAGTTCATTGATGATTTGCATAAGCTTGGCATATCTTGCCATTTTGAGAATGAAATCCTCCAAATCTTGAAATCCATATATCTTAATCAAAACTACAAAAGGGATTTGTACTCAACATCTCTAGCATTCAGACTCCTCAGACAATATGGCTTCATCCTTCCACAAGAAGTATTTGATTGTTTCAAGAATGAGGAGGGTACGGATTTCAAGCCAAGCTTCGGCCGTGATATCAAAGGCTTGTTACAATTGTATGAAGCTTCTTTCCTATCAAGAAAAGGAGAAGAAACTTTACAACTAGCAAGAGAGTTTGCAACAAAGATTCTGCAAAAAGAAGTTGATGAGAGAGAGTTTGCAACCAAGATGGAGTTCCCTTCTCATTGGACGGTTCAAATGCCGAATGCAAGACCTTTCATCGATGCTTACCGTAGGAGGCCGGATATGAATCCAGTTGTGCTCGAGCTAGCCATACTTGATACAAATATAGTTCAAGCACAATTTCAAGAAGAACTCAAAGAGACCTCAAGGTGGTGGGAGAGTACAGGCATTGTCCAAGAGCTTCCATTTGTGAGGGATAGGATTGTGGAAGGCTACTTTTGGACGATTGGAGTGACTCAGAGACGCGAGCATGGATACGAAAGAATCATGACCGCAAAGGTTATTGCCTTAGTAACATGTTTAGACGACATATACGATGTTTATGGCACGATAGAAGAGCTTCAACTTTTCACAAGCACAATCCAAAGATGGGATTTGGAATCAATGAAGCAACTCCCTACCTACATGCAAGTAAGCTTTCTTGCACTACACAACTTTGTAACCGAGGTGGCTTACGATACTCTCAAGAAAAAGGGCTACAACTCCACACCATATTTAAGAAAAACGTGGGTGGATCTTGTTGAATCATATATCAAAGAGGCAACTTGGTACTACAACGGTTATAAACCTAGTATGCAAGAATACCTTAACAATGCATGGATATCAGTCGGAAGTATGGCTATACTCAACCACCTCTTCTTCCGGTTCACAAACGAGAGAATGCATAAATACCGCGATATGAACCGTGTCTCGTCCAACATTGTGAGGCTTGCTGATGATATGGGAACATCATTGGCTGAGGTGGAGAGAGGGGACGTGCCGAAAGCAATTCAATGCTACATGAATGAGACGAATGCTTCTGAAGAAGAAGCAAGAGAATATGTAAGAAGAGTCATACAGGAAGAATGGGAAAAGTTGAACACAGAATTGATGCGGGATGATGATGATGATGATGATTTTACACTATCCAAATATTACTGTGAGGTGGTTGCTAATCTTACAAGAATGGCACAGTTTATATACCAAGATGGATCGGATGGCTTCGGCATGAAAGATTCCAAGGTTAATAGACTGCTAAAAGAGACGTTGATCGAGCGCTACGAATAACodon-optimized version of the L. angustifolia (La-S-β-PHLS) cDNAnucleotide sequence for expression in cyanobacteria, e.g., Synechocystis (“S” in gene designation). SEQ ID NO: 3TTAATTAACATATGTGTAGTTTGCAAGTTTCTGATCCTATTCCTACCGGACGCCGTTCCGGTGGTTATCCCCCGGCCTTATGGGATTTCGATACTATTCAATCCCTGAATACCGAATATAAGGGCGAACGTCACATGCGTCGGGAAGAAGACTTAATTGGTCAAGTTCGGGAAATGTTGGTGCACGAAGTAGAAGATCCCACTCCCCAGTTGGAATTCATTGACGATCTGCATAAATTGGGCATTTCCTGCCATTTTGAAAACGAGATTCTGCAAATTCTCAAATCCATTTATCTCAACCAAAACTATAAACGGGACCTCTATTCTACCAGTTTAGCCTTCCGTCTCTTGCGTCAATACGGGTTTATCTTGCCGCAGGAAGTTTTTGACTGCTTTAAAAACGAAGAAGGTACGGATTTTAAACCCAGCTTCGGCCGGGATATTAAGGGTCTGTTACAGTTGTACGAAGCCTCCTTTTTGTCCCGGAAGGGGGAAGAAACTTTACAACTCGCCCGCGAATTTGCTACCAAAATCTTGCAAAAGGAAGTCGATGAACGGGAATTTGCTACTAAAATGGAATTTCCCAGTCACTGGACCGTACAAATGCCTAACGCTCGGCCTTTTATCGATGCCTATCGTCGGCGTCCCGACATGAACCCCGTGGTTCTGGAACTCGCCATTCTCGATACCAATATCGTGCAAGCTCAGTTTCAAGAAGAATTGAAGGAGACCTCCCGTTGGTGGGAAAGCACGGGGATTGTTCAAGAACTGCCGTTTGTTCGGGACCGGATTGTGGAAGGTTATTTTTGGACCATTGGTGTTACTCAACGCCGTGAACACGGTTACGAACGTATTATGACGGCCAAAGTCATCGCTTTGGTGACCTGTTTGGATGATATTTATGACGTATATGGCACTATTGAAGAATTGCAACTCTTCACCTCTACGATTCAGCGTTGGGATTTGGAGTCTATGAAGCAGTTACCGACTTATATGCAGGTAAGCTTCCTGGCCTTGCACAATTTTGTAACCGAAGTGGCCTATGATACGCTGAAGAAAAAGGGCTACAACTCTACCCCCTATTTGCGGAAGACTTGGGTGGATTTGGTCGAAAGTTACATTAAGGAAGCCACTTGGTACTATAATGGGTACAAACCCTCTATGCAGGAATACCTCAACAACGCCTGGATCTCTGTGGGCAGCATGGCTATTTTGAATCATTTGTTTTTTCGCTTTACTAATGAACGCATGCATAAGTACCGGGACATGAATCGTGTATCCTCTAATATTGTGCGGTTAGCCGACGATATGGGAACCTCTTTGGCCGAAGTTGAACGCGGTGACGTGCCCAAAGCTATCCAATGTTACATGAATGAAACGAACGCCTCTGAGGAGGAGGCCCGCGAATATGTGCGGCGCGTTATCCAGGAAGAATGGGAAAAACTGAACACTGAACTGATGCGCGACGACGACGATGACGATGATTTCACCTTAAGTAAATACTACTGCGAAGTCGTTGCTAACCTGACCCGGATGGCTCAGTTCATTTACCAAGATGGTTCCGATGGGTTTGGGATGAAAGATTCCAAAGTAAATCGTTTACTGAAAGAAACGCTGATTGAGCGCTATGAGTGAAGATCTGCGGCC GC

What is claimed is:
 1. A method of obtaining β-phellandrene from photosynthetic microorganisms, the method comprising: culturing a strain of a photosynthetic microorganism that has been genetically modified to express a heterologous β-phellandrene synthase under conditions in which the β-phellandrene synthase is expressed; and collecting from the surface of the culture medium β-phellandrene hydrocarbons that have spontaneously diffused into the medium from the photosynthetic microorganisms across the cell wall, wherein the culture is in a continuous growth phase and the β-phellandrene hydrocarbons are continuously generated.
 2. The method of claim 1, wherein the strain of the photosynthetic microorganism is a cyanobacteria strain or a microalgae strain.
 3. The method of claim 1, wherein the strain of the photosynthetic microorganism is a cyanobacteria strain.
 4. The method of claim 3, wherein the cyanobacteria strain is from a genus selected from the group consisting of Synechocystis, Synechococcus, Arthrospira, Nostoc, and Anabaena.
 5. The method of claim 3, where the β-phellandrene synthase is encoded by a nucleic acid having at least 80% nucleic acid sequence identity to SEQ ID NO:3.
 6. The method of claim 1, wherein the strain of the photosynthetic microorganism is a microalgae strain.
 7. The method of claim 1, wherein the β-phellandrene synthase has at least 70% identity to SEQ ID NO:1.
 8. The method of claim 7, wherein the β-phellandrene synthase has at least 90% identity to SEQ ID NO:1.
 9. The method of claim 1, wherein collecting β-phellandrene hydrocarbons comprises siphoning or skimming the β-phellandrene hydrocarbons from the surface of the culture medium.
 10. The method of claim 1, wherein collecting β-phellandrene hydrocarbons comprises overlaying a solvent onto the surface of the culture medium.
 11. The method of claim 10, wherein the solvent is selected from the group consisting of heptane, decane, and dodecane.
 12. A cell culture comprising a photosynthetic microorganism, wherein the photosynthetic microorganism is genetically modified to express a heterologous β-phellandrene synthase; and cell culture media comprises β-phellandrene produced by the photosynthetic microorganism that has diffused into the cell culture media from the photosynthetic microorganism and floats on the surface of the culture medium, wherein the cell culture is in a continuous growth phase and the β-phellandrene hydrocarbons are continuously generated.
 13. The cell culture of claim 12, wherein the photosynthetic microorganism strain is a cyanobacteria strain or a microalgae strain.
 14. The cell culture of claim 12, wherein the photosynthetic microorganism strain is a cyanobacteria strain.
 15. The cell culture of claim 14, wherein the cyanobacteria strain is of a genus selected from the group consisting of the genera Synechocystis, Synechococcus, Arthrospira, Nostoc, and Anabaena. 